Regulated transcription of targeted genes and other biological events

ABSTRACT

Dimerization and oligomerization of proteins are general biological control mechanisms that contribute to the activation of cell membrane receptors, transcription factors, vesicle fusion proteins, and other classes of intra- and extracellular proteins. We have developed a general procedure for the regulated (inducible) dimerization or oligomerization of intracellular proteins. In principle, any two target proteins can be induced to associate by treating the cells or organisms that harbor them with cell permeable, synthetic ligands. To illustrate the practice of this invention, we have induced: (1) the intracellular aggregation of the cytoplasmic tail of the ζ chain of the T cell receptor (TCR)CD3 complex thereby leading to signaling and transcription of a reporter gene, (2) the homodimerization of the cytoplasmic tail of the Fas receptor thereby leading to cell-specific apoptosis (programmed cell death) and (3) the heterodimerization of a DNA-binding domain (Gal4) and a transcription-activation domain (VP16) thereby leading to direct transcription of a reporter gene. Regulated intracellular protein association with our cell permeable, synthetic ligands offers new capabilities in biological research and medicine, in particular, in gene therapy. Using gene transfer techniques to introduce our artificial receptors, one can turn on or off the signaling pathways that lead to the overexpression of therapeutic proteins by administering orally active &#34;dimerizers&#34; or &#34;de-dimerizers&#34;, respectively. Since cells from different recipients can be configured to have the pathway overexpress different therapeutic proteins for use in a variety of disorders, the dimerizers have the potential to serve as &#34;universal drugs&#34;. They can also be viewed as cell permeable, organic replacements for therapeutic antisense agents or for proteins that would otherwise require intravenous injection or intracellular expression (e.g., the LDL receptor or the CFTR protein).

STATEMENTS OF RIGHTS

This invention was made in the course of work supported by the U.S.Government. The U.S. Government therefore has certain rights in theinvention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.08/388,653, filed Feb. 14, 1995, U.S. Pat. No. 5,869,337 which is acontinuation-in-part of U.S. application Ser. No. 08/196,043, filed Feb.11, 1994, which in turn is a continuation-in-part of U.S. applicationSer. No. 08/179,748, filed Jan. 7, 1994, abandoned, which in turn is acontinuation-in-part of U.S. application Ser. No. 08/092,977, filed Jul.16, 1993, abandoned, which in turn is a continuation-in-part of U.S.application Ser. No. 08/017,931, filed Feb. 12, 1993, abandoned; and isa continuation-in-part of U.S. application Ser. No. 08/292,597, filedAug. 18, 1994, U.S. Pat. No. 5,834,266 which in turn is acontinuation-in-part of U.S. application Ser. No. 08/179,143, filed Jan.7, 1994, abandoned, which in turn is a continuation-in-part of U.S. Ser.No. 08/093,499, filed Jul. 16, 1993 abandoned. The contents of each ofthese applications is hereby incorporated by referenced into the presentdisclosure. The full contents of related cases PCT/US94/01617,PCT/US/94/01660 and PCT/US/94/08008 are also incorporated by referenceinto the present disclosure.

TECHNICAL FIELD

This invention concerns materials, methods and applications relating tothe oligomerizing of chimeric proteins with a dimeric or multimeric,preferably non-peptidic, organic molecule. Aspects of the invention areexemplified by recombinant modifications of host cells and their use ingene therapy or other applications of inducible gene expression.

INTRODUCTION

Biological specificity usually results from highly specific interactionsamong proteins. This principle is exemplified by signal transduction,the process by which extracellular molecules influence intracellularevents Many pathways originate with the binding of extracellular ligandsto cell surface receptors. In many cases receptor dimerization leads totransphosphorylation and the recruitment of proteins that continue thesignaling cascade. The realization that membrane receptors could beactivated by homodimerization resulted from the observation thatreceptors could be activated by antibodies that cross linked tworeceptors. Subsequently, many receptors were found to share thoseproperties. The extracellular and transmembrane regions of manyreceptors are believed to function by bringing the cytoplasmic domainsof the receptors in close proximity by a ligand-dependent dimerizationor oligomerization, while the cytoplasmic domains of the receptor conveyspecific signals to internal compartments of the cell.

Others have investigated ligand-receptor interactions in differentsystems. For example, Clark, et al., Science (1992) 258, 123 describecytoplasmic effectors of the B-cell antigen receptor complex. Durand, etal., Mol. Cell. Biol. (1988) 8, 1715, Verweij, et al., J. Biol. Chem.(1990) 265, 15788 and Shaw, et al., Science (1988) 241, 202 report thatthe NF-AT-directed transcription is rigorously under the control of theantigen receptor. Inhibition of NF-AT-directed transcription bycyclosporin A and FK506 is reported by Emmel, et al., Science (1989)246, 1617 and Flanagan, et al., Nature (1991) 352, 803. Durand, et al.,Mol. Cell. Biol. (1988) 8, 1715 and Mattila, et al., EMBO J. (1990) 9,4425 describe the NF-AT binding sites. References describing the ζ chaininclude Orloff, et al., Nature (1990) 347, 189-191; Kinet, et al., Cell(1989) 57, 351-354; Weissman, et al., Proc. Natl. Acad. Sci. USA (1988)85, 9709-9713 and Larder, Nature (1989) 342, 803-805. A CD4immunoadhesin is described by Byrn, et al. Nature (1990) 344, 667-670. ACD8-ζ-fused protein is described by Irving, et al., Cell (1992) 64, 891.See also, Letourner and Klausner, Science (1992) 255, 79.

Illustrative articles describing transcriptional factor association withpromoter regions and the separate activation and DNA binding oftranscription factors include: Keegan et al., Nature (1986) 231, 699;Fields and Song, ibid (1989) 340, 245; Jones, Cell (1990) 61, 9; Lewin,Cell (1990) 61, 1161; Ptashne and Gann, Nature (1990) 346, 329; Adamsand Workman, Cell (1993) 72, 306.

Illustrative articles describing vesicle targeting and fusion include:Sollner et al. (1993) Nature 362, 318-324; and Bennett and Scheller(1993) Proc. Natl. Acad. Sci. USA 90, 2559-2563.

Illustrative articles describing regulated protein degradation include:Hochstrasser et al (1990) Cell 61, 697; Scheffner, M. et al (1993) Cell75, 495; Rogers et al (1986) Science 234, 364-368.

Illustrative publications providing additional information concerningsynthetic techniques and modifications relevant to FK506 and relatedcompounds include: GB 2 244 991 A; EP 0 455 427 A1; WO 91/17754; EP 0465 426 A1, U.S. Pat. No. 5,023,263 and WO 92/00278.

However, as will be clear from this disclosure, none of the foregoingauthors describe or suggest the present invention. Our invention, whichis disclosed in detail hereinafter, involves a generally applicablemethod and materials for utilizing protein homodimerization,heterodimerization and oligomerization in living cells. Chimericresponder proteins are intracellularly expressed as fusion proteins witha specific receptor domain. Treatment of the cells with a cellpermeable. Multivalent ligand reagent which binds to the receptor domainleads to dimerization or oligomerization of the chimera. In analogy toother chimeric receptors (see e.g. Weiss, Cell (1993) 73,209), thechimeric proteins are designed such that oligomerization triggers thedesired subsequent events, e.g. the propagation of an intracellularsignal via subsequent protein--protein interactions and thereby theactivation of a specific subset of transcription factors. The initiationof transcription can be detected using a reporter gene assay.Intracellular crosslinking of chimeric proteins by synthetic ligands haspotential in basic investigation of a variety of cellular processes andin regulating the synthesis of proteins of therapeutic or agriculturalimportance. Furthermore, ligand mediated oligomerization now permitsregulated gene therapy. In so doing, it provides a fresh approach toincreasing the safety, expression level and overall efficacy obtainedwith gene therapy.

SUMMARY OF THE INVENTION

This invention provides novel chimeric (or "fused") proteins and smallorganic molecules capable of oligomerizing the chimeric proteins. Thechimeric proteins contain at least one ligand-binding (or "receptor")domain fused to an additional ("action") domain, as described in detailbelow. As will also be described, the chimeric proteins may containadditional domains as well. These chimeric proteins are recombinant inthe sense that the various domains are derived from different sources,and as such, are not found together in nature (i.e., are heterologous).

Genes, i.e., RNA or preferably DNA molecules referred to herein as"genetic" or "DNA" constructs) which encode the novel chimeric proteins,and optionally target genes, are provided for the genetic engineering ofhost cells. Also provided are methods and compositions for producing andusing such modified cells. The engineered cells of this inventioncontain at least one such chimeric protein or a first series of geneticconstructs encoding the chimeric protein(s). These constructs arerecombinant in the sense that the component portions, e.g. encoding aparticular domain or expression control sequence, are not found directlylinked to one another in nature (i.e., are heterologous).

One DNA construct of this invention encodes a chimeric proteincomprising (a) at least one receptor domain (capable of binding to aselected ligand) fused to (b) a heterologous additional ("action")protein domain. Significantly, the ligand is capable of binding to two(or more) receptor domains, i.e. to chimeric proteins containing suchreceptor domains, in either order or simultaneously, preferably with aKd value below about 10⁻⁶, more preferably below about 10⁻⁷, even morepreferably below about 10⁻⁸, and in some embodiments below about 10⁹⁻ M.The ligand preferably is a non-protein and has a molecular weight ofless than about 5 kDa. The receptor domains of the chimeric proteins sooligomerized maybe the same or different. The chimeric proteins arecapable of initiating a biological process upon exposure to the ligand,i.e., upon oligomerization with each other. The encoded chimeric proteinmay further comprises an intracellular targeting domain capable ofdirecting the chimeric protein to a desired cellular compartment. Thetargeting domain can be a secretory leader sequence, a membrane spanningdomain, a membrane binding domain or a sequence directing the protein toassociate with vesicles or with the nucleus, for instance.

The action domains of the chimeric proteins may be selected from a broadvariety of protein domains capable of effecting a desired biologicalresult upon oligomerization of the chimeric protein(s). For instance,the action domain may comprise a protein domain such as a CD3 zetasubunit capable, upon exposure to the ligand and subsequentoligomerization, of initiating a detectable intracellular signal; aDNA-binding protein such as Gal 4; or a transcriptional activationdomain such as VP16. Numerous other examples are provided herein. Oneexample of a detectable intracellular signal is a signal activating thetranscription of a gene under the transcriptional control of atranscriptional control element (e.g. enhancer/promoter elements and thelike) which is responsive to the oligomerization.

As is discussed in greater detail later, in various embodiments of thisinvention the chimeric protein is capable of binding to an FK506-typeligand, a cyclosporin A-type ligand, tetracycline or a steroid ligand.Such binding leads to oligomerization of the chimeric protein with otherchimeric protein molecules which may be the same or different.

Optionally the cells further contain a second recombinant geneticconstruct, or second series of such construct(s), containing a targetgene under the transcriptional control of a transcriptional controlelement (e.g. promoter/enhancer) responsive to a signal triggered byligand-mediated oligomerization of the chimeric proteins, i.e. toexposure to the ligand. These constructs are recombinant in the sensethat the target gene is not naturally under the transcriptional controlof the responsive transcriptional control element.

In one aspect of the invention the DNA construct contains (a) atranscriptional control element responsive to the oligomerization of achimeric protein as described above, and (b) flanking DNA sequence froma target gene permitting the homologous recombination of thetranscriptional control element into a host cell in association with thetarget gene. In other embodiments the construct contains a desired geneand flanking DNA sequence from a target locus permitting the homologousrecombination of the target gene into the desired locus. The constructmay also contain the responsive transcriptional control element, or theresponsive element may be provided by the locus. The target gene mayencodes a surface membrane protein, a secreted protein, a cytoplasmicprotein or a ribozyme or an antisense sequence.

The constructs of this invention may also contain a selectable markerpermitting transfection of the constructs into host cells and selectionof transfectants containing the construct. This invention furtherencompasses DNA vectors containing such constructs, whether for episomaltransfection or for integration into the host cell chromosomes. Thevetor may be a viral vector, including for example an adeno-, adenoassociated- or retroviral vector.

This invention further encompasses a chimeric protein encoded by any ofour DNA constructs, as well as cells containing and/or expressing them,including procaryotic and eucaryotic cells and in particular, yeast,worm, insect, mouse or other rodent, and other mammalian cells,including human cells, of various types and lineages, whether frozen orin active growth, whether in culture or in a whole organism containingthem.

For example, in one aspect, this invention provides cells, preferablybut not necessarily mammalian, which contain a first DNA constructencoding a chimeric protein comprising (i) at least one receptor domaincapable of binding to a selected oligomerizing ligand of this inventionand (ii) another protein domain, heterologous with respect to thereceptor domain, but capable, upon oligomerization with one or moreother like domains, of triggering the activation of transcription of atarget gene under the transcriptional control of a transcriptionalcontrol element responsive to said oligomerization. The cells furthercontain a target gene under the expression control of a transcriptionalcontrol element responsive to said oligomerization ligand. Followingexposure to the selected ligand expresses the target gene.

In another aspect, the invention provides cells which contain a firstset of DNA constructs encoding a first chimeric protein containing aDNA-binding domain and at least one receptor domain capable of bindingto a first selected ligand moiety. The cell further a second chimericprotein containing a transcriptional activating domain and at least onereceptor domain capable of binding to a second selected ligand (whichmaybe the same or different from the first selected ligand moiety). Thecell additional contains a DNA construct encoding a target gene underthe transcriptional control of a heterologous transcriptional controlsequence which binds with the DNA-binding domain and is responsive tothe transcriptional activating domain such that the cell expresses thetarget gene following exposure to a substance containing the selectedligand moiety(ies).

Also provided are ADNA composition comprising a first DNA constructencoding a chimeric protein comprising at least one receptor domain,capable of binding to a selected ligand, fused to a heterologousadditional protein domain capable of initiating a biological processupon exposure to the oligomerizing ligand, i.e. upon oligomerization ofthe chimeric protein; and a second DNA construct encoding a target geneunder the transcriptional control of a transcription control elementresponsive to the oligomerization ligand.

Another exemplary DNA composition of this invention comprises a firstseries of DNA constructs encoding a first and second chimeric proteinand a second DNA construct encoding a target gene under thetranscriptional control of an transcription control element responsiveto the oligomerization of the chimeric protein molecules. The DNAconstruct encoding the first chimeric protein comprises (a) at least onefirst receptor domain, capable of binding to a selected first ligandmoiety, fused to (b) a heterologous additional protein domain capable ofinitiating a biological process upon [exposure to the oligomerizationligand, i.e. upon oligomerization of the first chimeric protein to asecond chimeric protein molecule. The DNA construct encoding the secondchimeric protein comprises (i) at least one receptor domain, capable ofbinding to a selected second ligand moiety, fused to (ii) a heterologousadditional protein domain capable of initiating a biological processupon exposure to the oligomerization ligand, i.e., upon oligomerizationto the first chimeric protein. The first and second receptor moieties insuch cases may be the same or different and the first and secondselected ligand moieties may likewise be the same or different.

Our ligands are molecules capable of binding to two or more chimericprotein molecules of this invention to form an oligomer thereof, andhave the formula:

    linker--{rbm.sub.1, rbm.sub.2, . . . rbm.sub.n }

wherein n is an integer from 2 to about 5, rbm.sub.(1)- rbm.sub.(n) arereceptor binding moieties which may be the same or different and whichare capable of binding to the chimeric protein(s). The rbm moieties arecovalently attached to a linker moiety which is a bi- ormulti-functional molecule capable of being covalently linked ("--") totwo or more rbm moieties. Preferably the ligand has a molecular weightof less than about 5 kDa and is not a protein. Examples of such ligandsinclude those in which the rbm moieties are the same or different andcomprise an FK506-type moiety, a cyclosporin-type moiety, a steroid ortetracycline. Cyclosporin-type moieties include cyclosporin andderivatives thereof which are capable of binding to a cyclophilin,naturally occurring or modified, preferably with a Kd value below about10⁻⁶ M. In some embodiments it is preferred that the ligand bind to anaturally occurring receptor with a Kd value greater than about 10⁻⁶ Mand more preferably greater than about 10⁻⁵ M. Illustrative ligands ofthis invention are those in which at least one rbm comprises a moleculeof FK506, FK520, rapamycin or a derivative thereof modified at C9, C10or both, which ligands bind to a modified receptor or chimeric moleculecontaining a modified receptor domain with a Kd value at least one, andpreferably 2, and more preferably 3 and even more preferably 4 or 5 ormore orders of magnitude less than their Kd values with respect to anaturally occurring receptor protein. Linker moieties are also describedin detail later, but for the sake of illustration, include such moietiesas a C2-C20 alkylene, C4-C18 azalkylene, C6-C24 N-alkylene azalkylene,C6-18 arylene, C8-C24 ardialkylene or C8-C36 bis-carboxamido alkylenemoiety.

The monomeric rbm's of this invention, as well as compounds containingsole copies of an rbm, which are capable of binding to our chimericproteins but not effecting dimerization or higher order oligomerizationthereof (in view of the monomeric nature of the individual rbm) areoligomerization antagonists.

In one embodiment, genetically engineered cells of this invention can beused for regulated production of a desired protein. In that embodimentthe cells, engineered in accordance with this invention to express adesired gene under ligand-induced regulation, are grown in culture byconventional means. Addition of the ligand to the culture medium leadsto expression of the desired gene and production of the desired protein.Expression of the gene and production of the protein can then be turnedoff by adding to the medium an oligomerization antagonist reagent, as isdescribed in detail below. Alternatively, this invention can be used toengineer ligand-inducable cell death characteristics into cells. Suchengineered cells can then be eliminated from a cell culture after theyhave served their intended purposed (e.g. production of a desiredprotein or other product) by adding the ligand to the medium. Engineeredcells of this invention can also be used in vivo, to modify wholeorganisms, preferably animals, including humans, e.g. such that thecells produce a desired protein or other result within the animalcontaining such cells. Such uses include gene therapy. Alternatively,the chimeric proteins and oligomerizing molecules can be usedextracellularly to bring together proteins which act in concert toinitiate a physiological action.

This invention thus provides materials and methods for achieving abiological effect in cells in response to the addition of anoligomerizing ligand. The method involves providing cells engineered inaccordance with this invention and exposing the cells to the ligand.

For example, one embodiment of the invention is a method for activatingtranscription of a target gene in cells. The method involves providingcells containing and capable of expressing (a) at least one DNAconstruct encoding a chimeric protein of this invention and (b) a targetgene. The chimeric protein comprises at least one receptor domaincapable of binding to a selected oligomerization ligand. The receptordomain is fused to an action domain capable--upon exposure to theoligomerizing ligand, i.e., upon oligomerization with one or more otherchimeric proteins containing another copy of the action domain--ofinitiating an intracellular signal. That signal is capable of activatingtranscription of a gene, such as the target gene in this case, which isunder the transcriptional control of a transcriptional control elementresponsive to that signal. The method thus involves exposing the cellsto an oligomerization ligand capable of binding to the chimeric proteinin an amount effective to result in expression of the target gene. Incases in which the cells are growing in culture, exposing them to theligand is effected by adding the ligand to the culture medium. In casesin which the cells are present within a host organism, exposing them tothe ligand is effected by administering the ligand to the host organisimFor instance, in cases in which the host organism is an animal, inparticular, a mammal (including a human), the ligand is administered tothe host animal by oral, bucal, sublingual, transdermal, subcutaneous,intramuscular, intravenous, intra-joint or inhalation administration inan appropriate vehicle therefor.

This invention further encompasses a pharmaceutical compositioncomprising an oligomerization ligand of this invention in admixture witha pharmaceutically acceptable carrier and optionally with one or morepharmaceutically acceptable excipients for activating the transcriptionof a target gene, for example, or effecting another biological result ofthis invention, in a subject containing engineered cells of thisinvention. The oligomerization ligand can be a homo-oligomerizationreagent or a hetero-oligomerization reagent as described in detailelsewhere. Likewise, this invention further encompasses a pharmaceuticalcomposition comprising an oligomerization antagonist of this inventionadmixture with a pharmaceutically acceptable carrier and optionally withone or more pharmaceutically acceptable excipients for reducing, inwhole or part, the level of oligomerization of chimeric proteins inengineered cells of this invention in a subject, and thus forde-activating the transcription of a target gene, for example, orturning off another biological result of this invention. Thus, the useof the oligomerization reagents and of the oligomerization antagonistreagents to prepare pharmaceutical compositions is encompassed by thisinvention.

This invention also offers a method for providing a host organism,preferably an animal, and in many cases a mammal, responsive to anoligomerization ligand of this invention. The method involvesintroducing into the organism cells which have been engineered inaccordance with this invention, i.e. containing a DNA construct encodinga chimeric protein hereof, and so forth. Alternatively, one canintroduce the DNA constructs of this invention into a host organism,e.g. mammal under conditions permitting transfection of one or morecells of the host mammal.

We further provide kits for producing cells responsive to a ligand ofthis invention. One kit contains at least one DNA construct encoding oneof our chimeric proteins containing at least one receptor domain and anaction domain (as they are described elsewhere). The kit may contain aquantity of a ligand of this invention capable of oligomerizing thechimeric protein molecules encoded by the DNA constructs of the kit, andmay contain in addition a quantity of an oligomerization antagonist,e.g. monomeric ligand reagent. Where a sole chimeric protein is encodedby the construct(s), the oligomerization ligand is ahomo-oligomerization ligand. Where more than one such chimeric proteinis encoded, a hetero-oligomerization ligand may be included. The kit mayfurther contain a "second series" DNA construct encoding a target geneand/or transcription control element responsive to oligomerization ofthe chimeric protein molecules. The DNA constructs will preferably beassociated with one or more selection markers for convenient selectionof transfectants, as well as other conventional vector elements usefulfor replication in prokaryotes, for expression in a eukaryotes, and thelike. The selection markers may be the same or different for eachdifferent DNA construct, permitting the selection of cells which containeach such DNA construct(s).

For example, one kit of this invention contains a first DNA constructencoding a chimeric protein containing at least one receptor domain(capable of binding to a selected ligand), fused to a transcriptionalactivator domain; a second DNA construct encoding a second chimericprotein containing at least one receptor domain (capable of binding to aselected ligand), fused to a DNA binding domain; and a third DNAconstruct encoding a target gene under the control of a transcriptionalcontrol element containing a DNA sequence to which the DNA bindingdomain binds and which is transcriptionally activated by exposure to theligand in the presence of the first and second chimeric proteins.

Alternatively, a DNA construct for introducing a target gene under thecontrol of a responsive transcriptional control element may contain acloning site in place of a target gene to provide a kit for engineeringcells to inducably express a gene to be provided by the practitioner.

Other kits of this invention may contain one or two (or more) DNAconstructs for chimeric proteins in which one or more contain a cloningsite in place of an action domain (transcriptional initiation signalgenerator, transcriptional activator, DNA binding protein, etc.),permitting the user to insert whichever action domain she wishes. Such akit may optionally include other elements as described above, e.g. DNAconstruct for a target gene under responsive expression control,oligomerization ligand, antagonist, etc.

Any of the kits may also contain positive control cells which werestably transformed with constructs of this invention such that theyexpress a reporter gene (for CAT, beta-galactosidase or any convenientlydetectable gene product) in response to exposure of the cells to theligand. Reagents for detecting and/or quantifying the expression of thereporter gene may also be provided.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram of the plasmid pSXNeo/IL2 (IL2-SX). In NF-AT-SX, theHindIII-ClaI DNA fragment from IL2-SX containing the IL2enhancer/promoter, is replaced by a minimal IL-2 promoter conferringbasal transcription and an inducible element containing three tandemNFAT-finding sites (described below).

FIG. 2 is a flow diagram of the preparation of the intracellularsignaling chimera plasmids p#MXFn and p#MFnZ, where n indicates thenumber of binding domains.

FIGS. 3A and 3B are a flow diagram of the preparation of theextracellular signalling chimera plasmid p#1FK3/pBJ5.

FIGS. 4A, 4B and 4C are sequences of the primers used in theconstructions of the plasmids employed in the subject invention [SEQ IDNOS:4-40].

FIG. 5 is a chart of the response of reporter constructs havingdifferent enhancer groups to reaction of the receptor TAC/CD3ζ with aligand.

FIGS. 6A and 6B is a chart of the activity of various ligands with theTAg Jurkat cells described in Example 1.

FIG. 7 is a chart of the activity of the ligand FK1012A (8, FIG. 9B)with the extracellular receptor 1FK3 (FKBP×3/CD3 ζ).

FIG. 8 is a chart of the activation of an NFAT reporter via signallingthrough a myristoylated CD3 ζ/FKBP12 chimera.

FIG. 9A are the chemical structures of the allyl-linked FK506 variantsand the cyclohexyl-linked FK506 variants, respectively.

FIG. 10 is a flow diagram of the synthesis of derivatives of FK520.

FIGS. 11A, 11B, and 11C are a flow diagram of a synthesis of derivativesof FK520 and chemical structures of FK520, where the bottom structuresare designed to bind to mutant FKBP12.

FIG. 12 is a diagrammatic depiction of mutant FKBP with a modified FK520in the putative cleft.

FIGS. 13A and 13B is a flow diagram of the synthesis of heterodimers ofFK520 and cyclosporin.

FIG. 14 is a schematic representation of the oligomerization of chimericproteins, illustrated by chimeric proteins containing an immunophilinmoiety as the receptor domain.

FIG. 15 depicts ligand-mediated oligomerization of chimeric proteins,showing schematically the triggering of a transcriptional initiationsignal.

FIGS. 16A and 16B depicts synthetic schemes for HED and HOD reagentsbased on FK506-type moieties.

FIG. 17 depicts the synthesis of (CsA)2 beginning with CsA.

FIGS. 18A and 18B is an overview of the fusion cDNA construct andprotein MZF3E.

FIG. 19 depicts co-immunoprecipitation of MZF1E_(h) with MZFlE_(f) inthe presence of FK1012 (E_(h) : Flu-epitop-tag, E_(f) :Flag-epitop-tag).

FIG. 20 shows FK1012-induced cell death of the Jurkat T-cell linetransfected with a myristoylated Fas-FKBP12 fusion protein (MFF3E), asindicated by the decreased transcriptional activity of the cells.

FIG. 21A is an analysis of cyclophilin-Fas (and Fas-cyclophilin) fusionconstructs in the transient transfection assay. MC3FE was shown to bethe most effective in this series.

FIG. 21B depicts Immunphilin-Fas antigen chimeras and results oftransient expression experiments in Jurkat T cells stably transformedwith large T-antigen. Myr: the myristylation sequence taken frompp60^(c-src) encoding residues 1-14 (Wilson et al, Mol & Cell Biol 9 4(1989): 1536-44); FKBP: human FKBP12; CypC: murine cyclophilin Csequence encoding residues 36-212 (Freidman et al, Cell 66 4 (1991):799-806); Fas: intracellular domain of human Fas antigen encodingresidues 179-319 (Oehm et al, J Biol Chem 267 15 (1992): 10709-15).Cells were electroporated with a plasmid encoding a secreted alkalinephosphatase reporter gene under the control of 3 tandem AP1 promotersalong with a six fold molar excess of the immunophilin fusion construct.After 24 h the cells were stimulated with PMA (50 ng/mL), whichstimulates the synthesis of the reporter gene, and (CsA)2. At 48 h thecells were assayed for reporter gene activity. Western blots wereperformed at 24 hours anti-HA epitope antibodies.

FIG. 22 depicts CAT assay results from Example 8.

FIGS. 23A and 23B depicts the synthesis of modified FK-506 typecompounds.

DESCRIPTION

I. Generic Discussion

This invention provides chimeric proteins, organic molecules foroligomerizing the chimeric proteins and a system for using them. Thefused proteins have a binding domain for binding to the (preferablysmall) organic oligomerizing molecules and an action domain, which caneffectuate a physiological action or cellular process as a result ofoligomerization of the chimeric proteins.

The basic concept for inducible protein association is illustrated inFIG. 14. Ligands which can function as heterodimerization (orhetero-oligomerization, "HED") and homodimerization (orhomo-oligomerization, "HOD") agents are depicted as dumbell-shapedstructures.

(Homodimerization and homo-oligomerization refer to the association oflike components to form dimers or oligomers, linked as they are by theligands of invention. Heterodimerization and hetero-oligomerizationrefer to the association of dissimilar components to form dimers oroligomers. Homo-oligomers thus comprise an association of multiplecopies of a particular component while hetero-oligomers comprise anassociation of copies of different components. "Oligomerization","oligomerize" and "oligomer", as the terms are used herein, with orwithout prefixes, are intended to encompass "dimerization", "dimerize"and "dimer", absent an explicit indication to the contrary.)

Also depicted in FIG. 14 are fusion protein molecules containing atarget protein domain of interest ("action domain") and one or morereceptor domains that can bind to the ligands. For intracellularchimeric proteins, ie., proteins which are located within the cells inwhich they are produced, a cellular targeting sequence (includingorganelle targeting amino acid sequences) will preferably also bepresent. Binding of the ligand to the receptor domains hetero- orhomodimerizes the fusion proteins. Oligomerization brings the actiondomains into close proximity with one another thus triggering cellularprocesses normally associated with the respective action domain--such asTCR-mediated signal transduction, for example.

Cellular processes which can be triggered by oligomerization include achange in state, such as a physical state, e.g. conformational change,change in binding partner, cell death, initiation of transcription,channel opening, ion release, e.g. Ca⁺² etc. or a chemical state, suchas a chemical reaction, e.g. acylation, methylation, hydrolysis,phosphorylation or dephosphorylation, change in redox state,rearrangement, or the like. Thus, any such process which can betriggered by ligand-mediated oligomerization is included within thescope of this invention.

As a first application of the subject invention, cells are modified soas to be responsive to the oligomerizing molecules. The modified cellscan be used in gene therapy, as well as in other applications whereinducible transcription or translation (both are included under the termexpression) is desired. The cells are characterized by a genomecontaining at least a first or first series (the series may include onlyone construct) of genetic constructs, and desirably a second or secondseries (the series may include only one construct) of constructs.

The nature and number of such genetic constructs will depend on thenature of the chimeric protein and the role it plays in the cell. Forinstance, in embodiments where the chimeric protein is to be associatedwith expression of a gene (and which may contain an intracellulartargeting sequence or domain which directs the chimeric protein to beassociated with the cellular surface membrane or with an organelle e.g.nucleus or vesicle), then there will normally be at least two series ofconstructs: a first series encoding the chimeric protein(s) which uponligand-mediated oligomerization initiate a signal directing target geneexpression, and desirably a second series which comprise the target geneand/or expression control elements therefor which are responsive to thesignal.

Only a single construct in the first series will be required where ahomooligomer, usually a homodimer, is involved, while two or more,usually not more than three constructs may be involved, where aheterooligomer is involved. The chimeric proteins encoded by the firstseries of constructs will be associated with actuation of genetranscription and will normally be directed to the surface membrane orthe nucleus, where the oligomerized chimeric protein is able toinitiate, directly or indirectly, the transcription of one or moretarget genes. A second series of additional constructs will be requiredwhere an exogenous gene(s) is introduced, or where an exogenous orrecombinant expression control sequence is introduced (e.g. byhomologous recombination) for expression of an endogenous gene, ineither case, whose transcription will be activated by the oligomerizingof the chimeric protein.

A different first series of constructs are employed where the chimericproteins are intracellular and can act directly without initiation oftranscription of another gene. For example, proteins associated withexocytosis can be expressed inducibly or constitutively, where theproteins will not normally complex except in the presence of theoligomerizing molecule. By employing proteins which have any or all ofthese properties which do not complex in the host cell; are inhibited bycomplexation with other proteins, which inhibition may be overcome byoligomerization with the ligand; require activation through a processwhich is not available in the host cell; or by modifying the proteinswhich direct fusion of a vesicle with the plasma membrane to formchimeric proteins, where the extent of complex formation and membranefusion is enhanced in the presence of the oligomerizing molecule,exocytosis is or has the ability to be induced by the oligomerizingmolecule.

Other intracellular proteins, such as kinases, phosphatases and cellcycle control proteins can be similarly modified and used.

Various classes of genetic constructs of this invention are described asfollows:

(1) constructs which encode a chimeric protein comprising a bindingdomain and an action domain, where the binding domain is extracellularor intracellular and the action domain is intracellular such thatligand-mediated oligomerization of the chimeric protein, by itself (toform a homo-oligomer) or with a different fused protein comprising adifferent action domain (to form a hetero-oligomer), induces a signalwhich results in a series of events resulting in transcriptionalactivation of one or more genes;

(2) constructs which encode a chimeric protein having a binding domainand an action domain, where the binding domain and action domain are inthe nucleus, such that ligand-mediated oligomerization of the protein,by itself (to form a homo-oligomer) or with a different fused proteincomprising a different action domain (to form a hetero-oligomer),induces initiation of transcription directly via complexation of theoligomer(s) with the DNA transcriptional initiation region;

(3) constructs which encode a chimeric protein containing a bindingdomain and an action domain, where the binding domain and the actiondomain are cytoplasmic, such that ligand-mediated oligomerization of theprotein, by itself (to form a homo-oligomer) or with a different fusedprotein comprising a different action domain (to form ahetero-oligomer), results in exocytosis; and

(4) constructs which encode a chimeric protein containing a bindingdomain and an action domain, where the binding domain and action domainare extracellular and the action domain is associated with initiating abiological activity (by way of non-limiting illustration, the actiondomain can itself bind to a substance, receptor or other membraneprotein yielding, upon ligand-mediated oligomerization of the chimeras,the bridging of one or more similar or dissimilar molecules or cells);and,

(5) constructs which encode a destabilizing, inactivating or short-livedchimeric protein having a binding domain and an action domain, such thatligand-mediated oligomerization of the protein with a target proteincomprising a different action domain leads to the destabilization and/ordegradation or inactivation of said oligomerized target protein.

II. Transcription Regulation

The construct(s) of Groups (1) and (2), above, will be considered first.Group (1) constructs differ from group (2) constructs in their effect.Group (1) constructs are somewhat pleiotropic, i.e. capable ofactivating a number of wild-type genes, as well as the target gene(s).In addition, the response of the expression products of group (1) genesto the ligand is relatively slow. Group (2) constructs can be directedto a specific target gene and are capable of limiting the number ofgenes which will be transcribed. The response of expression products ofgroup (2) constructs to the ligand is very rapid.

The subject system for groups (1) and (2) will include a first series ofconstructs which comprise DNA sequences encoding the chimeric proteins,usually involving from one to three, usually one to two, differentconstructs. The system usually will also include a second series ofconstructs which will provide for expression of one or more genes,usually an exogenous gene. By "exogenous gene" is meant a gene which isnot otherwise normally expressed by the cell, e.g. because of the natureof the cell, because of a genetic defect of the cell, because the geneis from a different species or is a mutated or synthetic gene, or thelike. Such gene can encode a protein, antisense molecule, ribozyme etc.,or can be a DNA sequence comprising an expression control sequencelinked or to be linked to an endogenous gene with which the expressioncontrol sequence is not normally associated. Thus, as mentioned before,the construct can contain an exogenous or recombinant expression controlsequence for ligand-induced expression of an endogenous gene.

The chimeric protein encoded by a construct of groups (1), (2) and (3)can have, as is often preferred, an intracellular targeting domaincomprising a sequence which directs the chimeric protein to the desiredcompartment, e.g. surface membrane, nucleus, vesicular membrane, orother site, where a desired physiological activity can be initiated bythe ligand-mediated oligomerization, at least dimerization, of thechimeric protein.

The chimeric protein contains a second ("binding" or "receptor") domainwhich is capable of binding to at least one ligand molecule. Since theligand can contain more than one binding site or epitope, it can formdimers or higher order homo- or hetero-oligomers with the chimericproteins of this invention. The binding domain of the chimeric proteincan have one or a plurality of binding sites, so that homooligomers canbe formed with a divalent ligand. In this way the ligand can oligomerizethe chimeric protein by having two or more epitopes to which the seconddomain can bind, thus providing for higher order oligomerization of thechimeric protein.

The chimeric protein also contains a third ("action") domain capable ofinitiating a biological activity upon ligand-mediated oligomerization ofchimeric protein molecules via the binding domains. Thus, the actiondomain may be associated with transduction of a signal as a result ofthe ligand-mediated oligomerization. Such signal, for instance, couldresult in the initiation of transcription of one or more genes,depending on the particular intermediate components involved in thesignal transduction. See FIG. 15 which depicts an illustrative chimericprotein in which the intracellular tartgeting domain comprises amyristate moiety; the receptor domain comprises three FKBP12 moieties;and the action domain comprises a zeta subunit. In other chimericproteins the action domains may comprise transcription factors, whichupon oligomerization, result in the initiation of transcription of oneor more target genes, endogenous and/or exogenous. The action domainscan comprise proteins or portions thereof which are associated withfusion of vesicle membranes with the surface or other membrane, e.g.proteins of the SNAP and SNARE groups (See, Sollner et al. (1993) 362,318 and 353; Cell (1993) 72, 43).

A. Surface Membrane Receptor

Chimeric proteins of one aspect of this invention are involved with thesurface membrane and are capable of transducing a signal leading to thetranscription of one or more genes. The process involves a number ofauxiliary proteins in a series of interactions culminating in thebinding of transcription factors to promoter regions associated with thetarget gene(s). In cases in which the transcription factors bind topromoter regions associated with other genes, transcription is initiatedthere as well. A construct encoding a chimeric protein of thisembodiment can encode a signal sequence which can be subject toprocessing and therefore may not be present in the mature chimericprotein. The chimeric protein will in any event comprise (a) a bindingdomain capable of binding a predetermined ligand, (b) an optional(although in many embodiments, preferred) membrane binding domain whichincludes a transmembrane domain or an attached lipid for translocatingthe fused protein to the cell surface/membrane and retaining the proteinbound to the cell surface membrane, and, (c) as the action domain, acytoplasmic signal initiation domain. The cytoplasmic signal initiationdomain is capable of initiating a signal which results in transcriptionof a gene having a recognition sequence for the initiated signal in thetranscriptional initiation region.

The gene whose expression is regulated by the signal from the chimericprotein is referred to herein as the "target" gene, whether it is anexogenous gene or an endogenous gene under the expression control of anendogenous or exogenous (or hybrid) expression control sequence. Themolecular portion of the chimeric protein which provides for binding toa membrane is also referred to as the "retention domain". Suitableretention domains include a moiety which binds directly to the lipidlayer of the membrane, such as through lipid participation in themembrane or extending through the membrane, or the like. In such casesthe protein becomes translocated to and bound to the membrane,particularly the cellular membrane, as depicted in FIG. 15.

B. Nuclear Transcription Factors

Another first construct encodes a chimeric protein containing a cellulartargeting sequence which provides for the protein to be translocated tothe nucleus. This ("signal consensus") sequence has a plurality of basicamino acids, referred to as a bipartite basic repeat (reviewed inGarcia-Bustos et al, Biochimica et Biophysica Acta (1991) 1071, 83-101).This sequence can appear in any portion of the molecule internal orproximal to the N- or C-terminus and results in the chimeric proteinbeing inside the nucleus. The practice of one embodiment of thisinvention will involve at least two ("first series") chimeric proteins:(1) one having an action domain which binds to the DNA of thetranscription initiation region associated with a target gene and (2) adifferent chimeric protein containing as an action domain, atranscriptional activation domain capable, in association with the DNAbinding domain of the first chimeric protein, of initiatingtranscription of a target gene. The two action domains or transcriptionfactors can be derived from the same or different protein molecules.

The transcription factors can be endogenous or exogenous to the cellularhost. If the transcription factors are exogenous, but functional withinthe host and can cooperate with the endogenous RNA polymerase (ratherthan requiring an exogenous RNA polymerase, for which a gene could beintroduced), then an exogenous promoter element functional with thefused transcription factors can be provided with a second construct forregulating transcription of the target gene. By this means theinitiation of transcription can be restricted to the gene(s) associatedwith the exogenous promoter region, i.e., the target gene(s).

A large number of transcription factors are known which require twosubunits for activity. Alternatively, in cases where a singletranscription factor can be divided into two separate functional domains(e.g. a transcriptional activator domain and a DNA-binding domain), sothat each domain is inactive by itself, but when brought together indose proximity, transcriptional activity is restored. Transcriptionfactors which can be used include yeast GALA, which can be divided intotwo domains as described by Fields and Song, supra. The authors use afusion of GAL4(1-147)-SNF1 and SNF4-GAL4(768-881), where the SNF1 and -4may be replaced by the subject binding proteins as binding domains.Combinations of GAL4 and VP16 or HNF-1 can be employed. Othertranscription factors are members of the Jun, Fos, and ATF/CREBfamilies, Oct1, Sp1, HNF-3, the steriod receptor superfamily, and thelike.

As an alternative to using the combination of a DNA binding domain and anaturally occurring activation domain or modified form thereof, theactivation domain may be replaced by one of the binding proteinsassociated with bridging between a transcriptional activation domain andan RNA polymerase, including but not limited to RNA polymerase II. Theseproteins include the proteins referred to as TAF's, the TFII proteins,particularly B and D, or the like. Thus, one can use any one orcombination of proteins, for example, fused proteins or binding motifsthereof, which serve in the bridge between the DNA binding protein andRNA polymerase and provide for initiation of transcription. Preferably,the protein closest to the RNA polymerase will be employed inconjunction with the DNA binding domain to provide for initiation oftranscription. If desired, the subject constructs can provide for threeor more, usually not more than about 4, proteins to be brought togetherto provide the transcription initiation complex.

Rather than have a transcriptional activation domain as an actiondomain, an inactivation domain, such as ssn-6/TUP-1 or Kruppel-familysuppressor domain, can be employed. In this manner, regulation resultsin turning off the transcription of a gene which is constitutivelyexpressed. For example, in the case of gene therapy one can provide forconstitutive expression of a hormone, such as growth hormone, bloodproteins, immunoglobulins, etc. By employing constructs encoding onechimeric protein containing a DNA binding domain joined to a ligandbinding domain and another chimeric protein containing an inactivationdomain joined to a ligand binding domain, the expression of the gene canbe inhibited via ligand-mediated oligomerization.

Constructs encoding a chimeric protein containing inter alia aligand-binding domain fused to a transcriptional activating domain orsubunit, transcriptional inactivating domain or DNA-binding domain aredesigned and assembled in the same manner as described for the otherconstructs. Frequently, the N-terminus of the transcription factor willbe bound to the C-terminus of the ligand-binding domain, although insome cases the reverse will be true, for example, where two individualdomains of a single transcription factor are divided between twodifferent chimeras.

III. Exocytosis

Another use for the ligand-mediated oligomerization mechanism isexocytosis, where export of a protein rather than transcription iscontrolled by the ligand. This can be used in conjunction with theexpression of one or more proteins of interest, as an alternative toproviding for secretion of the protein(s) of interest via a secretorysignal sequence. This embodiment involves two different firstconstructs. One construct encodes a chimeric protein which directs theprotein to the vesicle to be integrated into the vesicular membrane asdescribed by Sollner et al., supra. Proteins which may be used as thevesicle binding protein include VAMP (synaptobrevin), SNC2, rab3, SEC4,synaptotagmin, etc., individually or in combination. The cellularmembrane protein may include syntaxin, SS01, SS02, neurexin, etc.,individually or in combination. The other construct provides fortransport to the surface membrane and employs the myristoyl signalsequence, other plasma membrane targeting sequence (e.g. forprenylation) or transmembrane retention domain, as described above. Theencoded proteins are described in the above references and, all orfunctional part, may serve as the action domains. These constructs couldbe used in conjunction with the expression of an exogenous protein,properly encoded for transport to a vesicle or for an endocytoticendogenous protein, to enhance export of the endogenous protein.

Various mechanisms can be employed for exocytosis. Depending on the celltype and which protein is limiting for endocytosis in the cell, one ormore of the vesicle bound proteins or cellular proteins may be encodedby one or more constructs having a response element which is activatedby the ligand. Of particular interest is the combination of VAMP andsyntaxin. Alteratively, one can provide for constitutive expression ofnon-limiting proteins controlling exocytosis and provide for ligandregulated expression of the exocytosis limiting protein. Finally, onecan provide for constitutive expression of the chimeric proteinsassociated with exocytosis, so that exocytosis is controlled byoligomerizing the chimeric proteins with the ligand. By employingappropriate binding domains, one can provide for different chimericproteins to be oligomerized on the vesicle surface to form an activecomplex, and/or linking of the vesicle protein(s) with the cell membranesurface protein through the ligand. The chimeric proteins may notprovide for exocytosis in the absence of the ligand due to modificationsin the ligand which substantially reduce the binding affinity betweenthe proteins governing exocytosis, such as deletions, mutations, etc.These modifications can be readily determined by employing overlappingfragments of the individual proteins and determining which fragmentsretain activity. The fragments can be further modified by using alaninesubstitutions to determine the individual amino acids whichsubstantially affect binding. (Beohncke et al., J. Immunol. (1993) 150,331-341; Evavold et al., ibid (1992) 148, 347-353).

The proteins assembled in the lumen of the vesicle, as well as the fusedproteins associated with exocytosis can be expressed constitutively orinducibly, as described above. Depending on the purpose of theexocytosis, whether endogenous or exogenous proteins are involved,whether the proteins to be exported are expressed constitutively orinducibly, whether the same ligand can be used for initiatingtranscription of the fused proteins associated with exocytosis and theproteins to be exported, or whether the different proteins are to besubject to different inducible signals, may determine the manner inwhich expression is controlled. In one aspect, the exocytosis mechanismwould be the only event controlled by the ligand. In other aspects, bothexpression of at least one protein and exocytosis may be subject toligand control.

Various proteins may be modified by introduction of a cellular targetingsequence for translocation of the protein to a vesicle without loss ofthe physiological activity of the protein. By using exocytosis as thedelivery mechanism, relatively high dosages may be delivered within ashort period of time to produce a high localized level of the protein ora high concentration in the vascular system, depending on the nature ofthe host. Proteins of interest include e.g. insulin, tissue plasminogenactivator, cytokines, erythropoietin, colony stimulating factors, growthfactors, inflammatory peptides, cell migration factors.

Coding sequences for directing proteins to a vesicle are available fromthe vesicle binding proteins associated with exocytosis. See, forexample, Sollner, et al. supra.

Another use of the oligomerization mechanism is the control of proteindegradation or inactivation. For example, a relatively stable orlong-lived chimeric protein of this invention can be destabilized ortargeted for degradation by ligand-mediated oligomerization with adifferent chimeric protein of this invention which has a relativelyshort half-life or which otherwise destabilizes or targets the oligomerfor degradation. In this embodiment, ligand-mediated oligomerizationregulates biological functioning of a protein by conferring upon it intrans a shortened half-life. The latter chimeric protein may contain adomain targeting the protein to the lysosome or a domain rendering theprotein susceptible to proteolytic cleavage in the cytosol or nucleus ornon-lysosomal organelle.

The half-life of proteins within cells is determined by a number offactors which include the presence of short amino acid sequences withinsaid protein rich in the amino acid residues proline, glutamic acid,serine and threonine, hence "PEST", other sequences with similarfunction, protease sensitive cleavage sites and the state ofubiquitinization. Ubiquitinization is the modification of a protein byone or more units of the short polypeptide chain, ubiquitin, whichtargets proteins for degradation. The rate of ubiquitinzation ofproteins is considered to be determined primarily by the identity of theN-terminal amino acid of the processed protein and one or more uniquelysine residues near the amino terminus.

IV. Other Regulatory Systems

Other biological functions which can be controlled by oligomerization ofparticular activities associated with individual proteins are proteinkinase or phosphatase activity, reductase activity, cyclooxygenaseactivity, protease activity or any other enzymatic reaction dependent onsubunit association. Also, one may provide for association of G proteinswith a receptor protein associated with the cell cycle, e.g. cyclins andcdc kinases, multiunit detoxifying enzymes.

V. Components of Constructs

The second or additional constructs (target genes) associated with group(1) and (2) chimeric proteins comprise a transcriptional initiationregion having the indicated target recognition sequence or responsiveelement, so as to be responsive to signal initiation from the activatedreceptor or activated transcription factors resulting in at least onegene of interest being transcribed to a sequence(s) of interest, usuallymRNA, whose transcription and, as appropriate, translation may result inthe expression of a protein and/or the regulation of other genes, e.g.antisense, expression of transcriptional factors, expression of membranefusion proteins, etc.

For the different purposes and different sites, different bindingdomains and different cytoplasmic domains will be used. For chimericprotein receptors associated with the surface membrane, if theligand-binding domain is extracellular, the chimeric protein can bedesigned to contain an extracellular domain selected from a variety ofsurface membrane proteins. Similarly, different cytoplasmic orintracellular domains of the surface membrane proteins which are able totransduce a signal can be employed, depending on which endogenous genesare regulated by the cytoplasmic portion. Where the chimeric protein isinternal, internal to the surface membrane protein or associated with anorganelle, e.g. nucleus, vesicle, etc., the ligand-binding domainprotein will be restricted to domains which can bind molecules which cancross the surface membrane or other membrane, as appropriate. Therefore,these binding domains will generally bind to small naturally occurringor synthetic ligand molecules which do not involve proteins or nucleicacids.

A. Cytoplasmic domains

A chimeric protein receptor of Group (1) can contain a cytoplasmicdomain from one of the various cell surface membrane receptors,including muteins thereof, where the recognition sequence involved ininitiating transcription associated with the cytoplasmic domain is knownor a gene responsive to such sequence is known. Mutant receptors ofinterest will dissociate transcriptional activation of a target genefrom activation of genes which can be associated with harmful sideeffects, such as deregulated cell growth or inappropriate release ofcytokines. The receptor-associated cytoplasmic domains of particularinterest will have the following characteristics: receptor activationleads to initiation of transcription for relatively few (desirably fewerthan 100) and generally innocuous genes in the cellular host; the otherfactors necessary for transcription initated by receptor activation arepresent in the cellular host; genes which are activated other than thetarget genes will not affect the intended purpose for which these cellsare to be used; oligomerization of the cytoplasmic domain or otheravailable mechanism results in signal initiation; and joining of thecytoplasmic domain to a desired ligand-binding domain will not interferewith signalling. A number of different cytoplasmic domains are known.Many of these domains are tyrosine kinases or are complexed withtyrosine kinases, e.g. CD3 ζ, IL-2R, IL-3R, etc. For a review seeCantley, et al., Cell (1991) 64, 281. Tyrosine kinase receptors whichare activated by cross-linking; e.g. dimerization (based on nomenclaturefirst proposed by Yarden and Ulrich, Annu. Rev. Biochem. (1988) 57,443;include subclass I: EGF-R, ATR2/neu, HER2/neu, HER3/c-erbB-3, Xmrk;subclass II: insulin-R, IGF-1-R [insulin-like growth factor receptor],IRR; subclass III: PDGF-R-A, PDGF-R-B, CSF-1-R (M-CSF/c-Fms), c-kit,STK-1/Flk-2; and subclass IV: FGF-R, flg [acidic FGF], bek [basic FGF]);neurotrophic tryosine kinases: Trk family, includes NGF-R, Ror1,2.Receptors which associate with tyrosine kinases upon cross-linkmnginclude the CD3 ζ-family: CD3 ζ and CD3 η (found primarily in T cells,associates with Fyn); β and γ chains of Fc.sub.ε RI (found primarily inmast cells and basophils); γ chain of Fc.sub.γ RIII/CD16 (foundprimarily in macrophages, neutrophils and natural killer cells); CD3γ,-δ, and -ε (found primarily in T cells); Igα/MB-1 and Ig-β/B29 (foundprimarily in B cell). Many cytokine and growth factor receptorsassociate with common β subunits which interact with tyrosine kinasesand/or other signalling molecules and which can be used as cytoplasmicdomains in chimeric proteins of this invention. These include (1) thecommon β subunit shared by the GM-CSF, IL-3 and IL-5 receptors; (2) theβ chain gp130 associated with the IL-6, leukemia inhibitory factor(LIF), ciliary neurotrophic factor (CNTF), oncostatin M, and IL-11receptors; (3) the IL-2 receptor γ subunit associated also withreceptors for IL-4, IL-7 and IL-13 (and possibly IL-9); and (4) the βchain of the IL-2 receptor which is homologous to the cytoplasmic domainof the G-CSF receptor.

The interferon family of receptors which include interferons α/β and γ(which can activate one or more members of the JAK, Tyk family oftyrosine kinases) as well as the receptors for growth hormone,erythropoietin and prolactin (which also can activate JAK2) can also beused as sources for cytoplasmic domains.

Other sources of cytoplasmic domains include the TGF-β family of cellsurface receptors (reviewed by Kingsley, D., Genes and Development 19948 133). This family of receptors contains serine/threonine kinaseactivity in their cytoplasmic domains, which are believed to beactivated by crosslinking.

The tyrosine kinases associated with activation and inactivation oftranscription factors are of particular interest in providing specificpathways which can be controlled and can be used to initiate or inhibitexpression of an exogenous gene.

The following table provides a number of receptors and characteristicsassociated with the receptor and their nuclear response elements thatactivate genes. The list is not exhaustive, but provides exemplarysystems for use in the subject invention.

In many situations mutated cytoplasmic domains can be obtained where thesignal which is transduced may vary from the wild type, resulting in arestricted or different pathway as compared to the wild-type pathway(s).For example, in the case of growth factors, such as EGF and FGF,mutations have been reported where the signal is uncoupled from cellgrowth but is still maintained with c-fos (Peters, et al., Nature (1992)358, 678).

The tyrosine kinase receptors can be found on a wide variety of cellsthroughout the body. In contrast, the CD3 ζ-family, the Ig family andthe lymphokine β-chain receptor family are found primarily onhematopoietic cells, particularly T-cells, B-cells, mast cells,basophils, macrophages, neutrophils, and natural killer cells. Thesignals required for NF-AT transcription come primarily from the zeta(ζ) chain of the antigen receptor and to a lesser extent CD3γ,δ,ε.

                  TABLE 1                                                         ______________________________________                                               DNA      Binding                                                                                        Ligand Element Factor(s) Gene Reference      ______________________________________                                        Insulin                                                                              cAMP     LRFI     jun-B  Mol. Cell Biol. (1992),                         and others responsive  many 12, 4654                                           element  genes PNAS, 83, 3439                                                 (cre)                                                                        PDGF, SRE SRF/SR c-fos Mol. Cell Biol. (1992),                                FGF, TGF  EBP  12, 4769                                                       and others                                                                    EGF VL30  RVL-3 Mol. Cell. Biol. (1992),                                       RSRF  virus 12, 2793                                                            c-jun Mol. Cell. Biol. (1992),                                                 12, 4472                                                                  IFN-α ISRE ISGF-3  Gene Dev. (1989) 3,                                      1362                                                                      IFN-γ GAS GAF GBP Mol. Cel. Biol. (1991)                                    11, 182                                                                   PMA and  AP-1 many Cell (1987) 49, 729-739                                    TCR   genes                                                                   TNF  NFκB many Cell (1990) 62, 1019-                                       genes 1029                                                                 Antigen ARRE-1 OAP/O many Mol. Cell Biol. (1988)                                ct-1 genes 8, 1715                                                          Antigen ARRE-2 NFAT IL-2 Science (1988) 241, 202                                 enhancer                                                                 ______________________________________                                    

The cytoplasmic domain, as it exists naturally or as it may betruncated, modified or mutated, will be at least about 10, usually atleast about 30 amino adds, more usually at least about 50 amino acids,and generally not more than about 400 amino acids, usually not more thanabout 200 amino adds. (See Romeo, et al., Cell (1992) 68, 889-893.)While any species can be employed, the species endogenous to the hostcell is usually preferred. However, in many cases, the cytoplasmicdomain from a different species can be used effectively. Any of theabove indicated cytoplasmic domains may be used, as well as others whichare presently known or may subsequently be discovered.

For the most part, the other chimeric proteins associated withtranscription factors, will differ primarily in having a cellulartargeting sequence which directs the chimeric protein to the internalside of the nuclear membrane and having transcription factors orportions thereof as the action domains. Usually, the transcriptionfactor action domains can be divided into "DNA binding domains" and"activation domains." One can provide for a DNA binding domain with oneor more ligand binding domains and an activation domain with one or moreligand binding domains. In this way the DNA binding domain can becoupled to a plurality of binding domains and/or activation domains.Otherwise, the discussion for the chimeric proteins associated with thesurface membrane for signal transduction is applicable to the chimericproteins for direct binding to generic DNA. Similarly, the chimericprotein associated with exocytosis will differ primarily as to theproteins associated with fusion of the vesicle membrane with the surfacemembrane, in place of the transducing cytoplasmic proteins.

B. Cellular Targeting Domains

A signal peptide or sequence provides for transport of the chimericprotein to the cell surface membrane, where the same or other sequencescan encode binding of the chimeric protein to the cell surface membrane.While there is a general motif of signal sequences, two or threeN-terminal polar amino adds followed by about 15-20 primarilyhydrophobic amino adds, the individual amino acids can be widely varied.Therefore, substantially any signal peptide can be employed which isfunctional in the host and may or may not be associated with one of theother domains of the chimeric protein. Normally, the signal peptide isprocessed and will not be retained in the mature chimeric protein. Thesequence encoding the signal peptide is at the 5'-end of the codingsequence and will include the initiation methionine codon.

The choice of membrane retention domain is not critical to thisinvention, since it is found that such membrane retention domains aresubstantially fungible and there is no critical amino acid required forbinding or bonding to another membrane region for activation. Thus, themembrane retention domain can be isolated from any convenient surfacemembrane or cytoplasmic protein, whether endogenous to the host cell ornot.

There are at least two different membrane retention domains: atransmembrane retention domain, which is an amino acid sequence whichextends across the membrane; and a lipid membrane retention domain,which lipid associates with the lipids of the cell surface membrane.

For the most part, for ease of construction, the transmembrane domain ofthe cytoplasmic domain or the receptor domain can be employed, which maytend to simplify the construction of the fused protein. However, for thelipid membrane retention domain, the processing signal will usually beadded at the 5' end of the coding sequence for N-terminal binding to themembrane and, proximal to the 3' end for C-terminal binding. The lipidmembrane retention domain will have a lipid of from about 12 to 24carbon atoms, particularly 14 carbon atoms, more particularly myristoyl,joined to glycine. The signal sequence for the lipid binding domain isan N-terminal sequence and can be varied widely, usually having glycineat residue 2 and lysine or arginine at residue 7 (Kaplan, et al., Mol.Cell. Biol. (1988) 8, 2435). Peptide sequences involvingpost-translational processing to provide for lipid membrane binding aredescribed by Carr, et al., PNAS USA (1988) 79, 6128; Aitken, et al.,FEBS Lett. (1982) 150, 314; Henderson, et al., PNAS USA (1983) 80, 319;Schulz, et al., Virology (1984), 123, 2131; Dellman, et al., Nature(1985) 314, 374; and reviewed in Ann. Rev. of Biochem. (1988) 57,69. Anamino acid sequence of interest includes the sequenceM-S-S-K-S-K-P-K-D-P-S-Q-R [SEQ ID NO:1]. Various DNA sequences can beused to encode such sequence in the fused receptor protein.

Generally, the transmembrane domain will have from about 18-30 aminoacids, more usually about 20-30 amino acids, where the central portionwill be primarily neutral, non-polar amino acids, and the termini of thedomain will be polar amino acids, frequently charged amino acids,generally having about 1-2 charged, primarily basic amino acids at thetermini of the transmembrane domain followed by a helical break residue,e.g. pro- or gly-.

C. Ligand Binding Domain

The ligand binding ("dimerization") domain of a chimeric protein of thisinvention can be any convenient domain which will allow for inductionusing a natural or unnatural ligand, preferably an unnatural syntheticligand. The binding domain can be internal or external to the cellularmembrane, depending upon the nature of the construct and the choice ofligand. A wide variety of binding proteins, including receptors, areknown, including binding proteins associated with the cytoplasmicregions indicated above. Of particular interest are binding proteins forwhich ligands (preferably small organic ligands) are known or may bereadily produced. These receptors or ligand binding domains include theFKBPs and cyclophilin receptors, the steriod receptors, the tetracyclinereceptor, the other receptors indicated above, and the like, as well as"unnatural" receptors, which can be obtained from antibodies,particularly the heavy or light chain subunit, mutated sequencesthereof, random amino acid sequences obtained by stochastic procedures,combinatorial syntheses, and the like. For the most part, the receptordomains will be at least about 50 amino acids, and fewer than about 350amino acids, usually fewer than 200 amino acids, either as the naturaldomain or truncated active portion thereof. Preferably the bindingdomain will be small (<25 kDa, to allow efficient transfection in viralvectors), monomeric (this rules out the avidin-biotin system),nonimmunogenic, and should have synthetically accessible, cellpermeable, nontoxic ligands that can be configured for dimerization.

The receptor domain can be intracellular or extracellular depending uponthe design of the construct encoding the chimeric protein and theavailability of an appropriate ligand. For hydrophobic ligands, thebinding domain can be on either side of the membrane, but forhydrophilic ligands, particularly protein ligands, the binding domainwill usually be external to the cell membrane, unless there is atransport system for internalizing the ligand in a form in which it isavailable for binding. For an intracellular receptor, the construct canencode a signal peptide and transmembrane domain 5' or 3' of thereceptor domain sequence or by having a lipid attachment signal sequence5' of the receptor domain sequence. Where the receptor domain is betweenthe signal peptide and the transmembrane domain, the receptor domainwill be extracellular.

The portion of the construct encoding the receptor can be subjected tomutagenesis for a variety of reasons. The mutagenized protein canprovide for higher binding affinity, allow for discrimination by theligand of the naturally occurring receptor and the mutagenized receptor,provide opportunities to design a receptor-ligand pair, or the like. Thechange in the receptor can involve changes in amino acids known to be atthe binding site, random mutagenesis using combinatorial techniques,where the codons for the amino acids associated with the binding site orother amino acids associated with conformational changes can be subjectto mutagenesis by changing the codon(s) for the particular amino acid,either with known changes or randomly, expressing the resulting proteinsin an appropriate prokaryotic host and then screening the resultingproteins for binding. Illustrative of this situation is to modifyFKBP12's Phe36 to Ala and/or Asp37 to Gly or Ala to accommodate asubstituent at positions 9 or 10 of FK506 or FK520. In particular,mutant FKBP12 moieties which contain Val, Ala, Gly, Met or other smallamino acids in place of one or more of Tyr26, Phe36, Asp37, Tyr82 andPhe99 are of particular interest as receptor domains for FK506-type andFK-520-type ligands containing modifications at C9 and/or C10.

Antibody subunits, e.g. heavy or light chain, particularly fragments,more particularly all or part of the variable region, or fusions ofheavy and light chain to create high-affinity binding, can be used asthe binding domain. Antibodies can be prepared against haptenicmolecules which are physiologically acceptable and the individualantibody subunits screened for binding affinity. The cDNA encoding thesubunits can be isolated and modified by deletion of the constantregion, portions of the variable region, mutagenesis of the variableregion, or the like, to obtain a binding protein domain that has theappropriate affinity for the ligand. In this way, almost anyphysiologically acceptable haptenic compound can be employed as theligand or to provide an epitope for the ligand. Instead of antibodyunits, natural receptors can be employed, where the binding domain isknown and there is a useful ligand for binding.

The ability to employ in vitro mutagenesis or combinatorialmodifications of sequences encoding proteins allows for the productionof libraries of proteins which can be screened for binding affinity fordifferent ligands. For example, one can totally randomize a sequence of1 to 5, 10 or more codons, at one or more sites in a DNA sequenceencoding a binding protein, make an expression construct and introducethe expression construct into a unicellular microorganism, and develop alibrary. One can then screen the library for binding affinity to one ordesirably a plurality of ligands. The best affinity sequences which arecompatible with the cells into which they would be introduced can thenbe used as the binding domain. The ligand would be screened with thehost cells to be used to determine the level of binding of the ligand toendogenous proteins. A binding profile could be defined weighting theratio of binding affinity to the mutagenized binding domain with thebinding affinity to endogenous proteins. Those ligands which have thebest binding profile could then be used as the ligand. Phage displaytechniques, as a non-limiting example, can be used in carrying out theforegoing.

D. Multimerization

The transduced signal will normally result from ligand-mediatedoligomerization of the chimeric protein molecules, i.e. as a result ofoligomerization following ligand binding, although other binding events,for example allosteric activation, can be employed to initiate a signal.The construct of the chimeric protein will vary as to the order of thevarious domains and the number of repeats of an individual domain. Forthe extracellular receptor domain in the 5'-3' direction oftranscription, the construct will encode a protein comprising the signalpeptide, the receptor domain, the transmembrane domain and the signalinitiation domain, which last domain will be intracellular(cytoplasmic). However, where the receptor domain is intracellular,different orders may be employed, where the signal peptide can befollowed by either the receptor or signal initiation domain, followed bythe remaining domain, or with a plurality of receptor domains, thesignal initiation domain can be sandwiched between receptor domains.Usually, the active site of the signal initiation domain will beinternal to the sequence and not require a free carboxyl terminus.Either of the domains can be multimerized, particularly the receptordomain, usually having not more than about 5 repeats, more usually notmore than about 3 repeats.

For multimerizing the receptor, the ligand for the receptor domains ofthe chimeric surface membrane proteins will usually be multimeric in thesense that it will have at least two binding sites, with each of thebinding sites capable of binding to the receptor domain. Desirably, thesubject ligands will be a dimer or higher order oligomer, usually notgreater than about tetrameric, of small synthetic organic molecules, theindividual molecules typically being at least about 150 D and fewer thanabout 5 kD, usually fewer than about 3 kD. A variety of pairs ofsynthetic ligands and receptors can be employed. For example, inembodiments involving natural receptors, dimeric FK506 can be used withan FKBP receptor, dimerized cyclosporin A can be used with thecyclophilin receptor, dimerized estrogen with an estrogen receptor,dimerized glucocorticoids with a glucocorticoid receptor, dimerizedtetracycline with the tetracycline receptor, dimerized vitamin D withthe vitamin D receptor, and the like. Alternatively higher orders of theligands, e.g. trimeric can be used. For embodiments involving unnaturalreceptors, e.g. antibody subunits, modified antibody subunits ormodified receptors and the like, any of a large variety of compounds canbe used. A significant characteristic of these ligand units is that theybind the receptor with high affinity (preferably with a K_(d) ≦10⁻ M)and are able to be dimerized chemically.

The ligand can have different receptor binding molecules with differentepitopes (also referred to as "HED" reagents, since they can mediatehetero-dimerization or hetero-oligomerization of chimeric proteinshaving the same or different binding domains. For example, the ligandmay comprise FK506 or an FK506-type moiety and a CsA or a cyclosporintype moiety. Both moieties are covalently attached to a common linkermoiety. Such a ligand would be useful for mediating the oligomerizationof a first and second chimeric protein where the first chimeric proteincontains a receptor domain such as an FKBP12 which is capable of bindingto the FK506-type moiety and the second chimeric protein contains areceptor domain such as cyclophilin which is capable of binding to thecyclosporin A-type moiety.

VI. Cells

The cells may be procaryotic, but are preferably eucaryotic, includingplant, yeast, worm, insect and mammalian. At present it is especiallypreferred that the cells be mammalian cells, particularly primate, moreparticularly human, but can be associated with any animal of interest,particularly domesticated animals, such as equine, bovine, murine,ovine, canine, feline, etc. Among these species, various types of cellscan be involved, such as hematopoietic, neural, mesenchymal, cutaneous,mucosal, stromal, muscle, spleen, reticuloendothelial, epithelial,endothelial, hepatic, kidney, gastrointestinal, pulmonary, etc. Ofparticular interest are hematopoietic cells, which include any of thenucleated cells which may be involved with the lymphoid ormyelomonocytic lineages. Of particular interest are members of the T-and B-cell lineages, macrophages and monocytes, myoblasts andfibroblasts. Also of particular interest are stem and progenitor cells,such as hematopoietic neural, stromal, muscle, hepatic, pulmonary,gastrointestinal, etc.

The cells can be autologous cells, syngeneic cells, allogenic cells andeven in some cases, xenogeneic cells. The cells may be modified bychanging the major histocompatibility complex ("MHC") profile, byinactivating β₂ -microglobulin to prevent the formation of functionalClass I MHC molecules, inactivation of Class II molecules, providing forexpression of one or more MHC molecules, enhancing or inactivatingcytotoxic capabilities by enhancing or inhibiting the expression ofgenes associated with the cytotoxic activity, or the like.

In some instances specific clones or oligoclonal cells may be ofinterest, where the cells have a particular specificity, such as T cellsand B cells having a specific antigen specificity or homing target sitespecificity.

VII. Ligands

A wide variety of ligands, including both naturally occurring andsynthetic substances, can be used in this invention to effectoligomerization of the chimeric protein molecules. Applicable andreadily observable or measurable criteria for selecting a ligand are:(A) the ligand is physiologically acceptable (i.e., lacks undue toxicitytowards the cell or animal for which it is to be used), (B) it has areasonable therapeutic dosage range, (C) desirably (for applications inwhole animals, including gene therapy applications), it can be takenorally (is stable in the gastrointestinal system and absorbed into thevascular system), (D) it can cross the cellular and other membranes, asnecessary, and (E) binds to the receptor domain with reasonable affinityfor the desired application. A first desirable criterion is that thecompound is relatively physiologically inert, but for its activatingcapability with the receptors. The less the ligand binds to nativereceptors and the lower the proportion of total ligand which binds tonature receptors, the better the response will normally be.Particularly, the ligand should not have a strong biological effect onnative proteins. For the most part, the ligands will be non-peptide andnon-nucleic acid.

The subject compounds will for the most part have two or more units,where the units can be the same or different, joined together through acentral linking group. The "units" will be individual moieties (e.g.,FK506, FK520, cyclosporin A, a steroid, etc.) capable of binding thereceptor domain. Each of the units will usually be joined to the linkinggroup through the same reactive moieties, at least in homodimers orhigher order homo-oligomers.

As indicated above, there are a variety of naturally-occurring receptorsfor small non-proteinaceous organic molecules, which small organicmolecules fulfill the above criteria, and can be dimerized at varioussites to provide a ligand according to the subject invention.Substantial modifications of these compounds are permitted, so long asthe binding capability is retained and with the desired specificity.Many of the compounds will be macrocyclics, e.g. macrolides. Suitablebinding affinities will be reflected in Kd values well below 10⁻⁴preferably below 10⁻⁶, more preferably below about 10⁻⁷, althoughbinding affinities below 10⁻⁹ or 10⁻¹⁰ are possible, and in some caseswill be most desirable.

Currently preferred ligands comprise oligomers, usually dimers, ofcompounds capable of binding to an FKBP protein and/or to a cyclophilinprotein. Such ligands includes homo- and heteromultimers (usually 2-4,more usually 2-3 units) of cyclosporin A, FK506, FK520, and rapamycin,and derivatives thereof, which retain their binding capability to thenatural or mutagenized binding domain. Many derivatives of suchcompounds are already known, including synthetic high affinity FKBPligands, which can be used in the practice of this invention. See e.g.Holt et al, J Am Chem Soc 1993, 115, 9925-9935. Sites of interest forlinking of FK506 and analogs thereof include positions involving annularcarbon atoms from about 17 to 24 and substituent positions bound tothose annular atoms, e.g. 21 (allyl), 22, 37, 38, 39 and 40, or 32(cyclohexyl), while the same positions except for 21 are of interest forFK520. For cyclosporin, sites of interest include MeBmt, position 3 andposition 8.

Of particular interest are modifications to the ligand which change itsbinding characteristics, particularly with respect to the ligand'snaturally occurring receptor. Concomitantly, one would change thebinding protein to accommodate the change in the ligand. For example,one can modify the groups at position 9 or 10 of FK506 (see Van Duyne etal (1991) Science 252, 839), so as to increase their steric requirement,by replacing the hydroxyl with a group having greater stericrequirements, or by modifying the carbonyl at position 10, replacing thecarbonyl with a group having greater steric requirements orfunctionalizing the carbonyl, e.g. forming an N-substituted Schiff'sbase or imine, to enhance the bulk at that position. Variousfunctionalities which can be conveniently introduced at those sites arealkyl groups to form ethers, acylamido groups, N-alkylated amines, wherea 2-hydroxyethylimine can also form a 1,3-oxazoline, or the like.Generally, the substituents will be from about 1 to 6, usually 1 to 4,and more usually 1 to 3 carbon atoms, with from 1 to 3, usually 1 to 2heteroatoms, which will usually be oxygen, sulfur, nitrogen, or thelike. By using different derivatives of the basic structure, one cancreate different ligands with different conformational requirements forbinding. By mutagenizing receptors, one can have different receptors ofsubstantially the same sequence having different affinities for modifiedligands not differing significantly in structure.

Other ligands which can be used are steroids. The steroids can beoligomerized, so that their natural biological activity is substantiallydiminished without loss of their binding capability with respect to achimeric protein containing one or more steroid receptor domains. By wayof non-limiting example, glucocorticoids and estrogens can be so used.Various drugs can also be used, where the drug is known to bind to aparticular receptor with high affinity. This is particularly so wherethe binding domain of the receptor is known, thus permitting the use inchimeric proteins of this invention of only the binding domain, ratherthan the entire native receptor protein. For this purpose, enzymes andenzyme inhibitors can be used.

A. Linkers

Various functionalities can be involved in the linking, such as amidegroups, including carbonic acid derivatives, ethers, esters, includingorganic and inorganic esters, amino, or the like. To provide forlinking, the particular monomer can be modified by oxidation,hydroxylation, substitution, reduction, etc., to provide a site forcoupling. Depending on the monomer, various sites can be selected as thesite of coupling.

The multimeric ligands can be synthesized by any convenient means, wherethe linking group will be at a site which does not interfere with thebinding of the binding site of a Iigand to the receptor. Where theactive site for physiological activity and binding site of a ligand tothe receptor domain are different, it will usually be desirable to linkat the active site to inactivate the ligand. Various linking groups canbe employed, usually of from 1-30, more usually from about 1-20 atoms inthe chain between the two molecules (other than hydrogen), where thelinking groups will be primarily composed of carbon, hydrogen, nitrogen,oxygen, sulphur and phosphorous. The linking groups can involve a widevariety of functionalities, such as amides and esters, both organic andinorganic, amines, ethers, thioethers, disulfides, quaternary ammoniumsalts, hydrazines, etc. The chain can include aliphatic, alicyclic,aromatic or heterocyclic groups. The chain will be selected based onease of synthesis and the stability of the multimeric ligand. Thus, ifone wishes to maintain long-term activity, a relatively inert chain willbe used, so that the multimeric ligand link will not be cleaved.Alternatively, if one wishes only a short half-life in the blood stream,then various groups can be employed which are readily cleaved, such asesters and amides, particularly peptides, where circulating and/orintracellular proteases can cleave the linking group.

Various groups can be employed as the linking group between ligands,such as allylene, usually of from 2 to 20 carbon atoms, azalkylene(where the nitrogen will usually be between two carbon atoms), usuallyof from 4 to 18 carbon atoms), N-alkylene azalkylene (see above),usually of from 6 to 24 carbon atoms, arylene, usually of from 6 to 18carbon atoms, ardialkylene, usually of from 8 to 24 carbon atoms,bis-carboxamido alkylene of from about 8 to 36 carbon atoms, etc.Illustrative groups include decylene, octadecylene, 3-azapentylene,5-azadecylene, N-butylene 5-azanonylene, phenylene, xylylene,p-dipropylenebenzene, bis-benzoyl 1,8-diaminooctane and the like.Multivalent or other (see below) ligand molecules containing linkermoieties as described above can be evaluated with chimeric proteins ofthis invention bearing corresponding receptor domains using materialsand methods described in the examples which follow.

B. Ligand Characteristics

For intracellular binding domains, the ligand will be selected to beable to be transferred across the membrane in a bioactive form, that is,it will be membrane permeable. Various ligands are hydrophobic or can bemade so by appropriate modification with lipophilic groups.Particularly, the linking bridge can serve to enhance the lipophilicityof the ligand by providing aliphatic side chains of from about 12 to 24carbon atoms. Alternatively, one or more groups can be provided whichwill enhance transport across the membrane, desirably without endosomeformation.

In some instances, multimeric ligands need not be employed. For example,molecules can be employed where two different binding sites provide fordimerization of the receptor. In other instances, binding of the ligandcan result in a conformational change of the receptor domain, resultingin activation, e.g. oligomerization, of the receptor. Other mechanismsmay also be operative for inducing the signal, such as binding a singlereceptor with a change in conformation resulting in activation of thecytoplasmic domain.

C. Ligand Antagonists

Monomeric ligands can be used for reversing the effect of the multimericligand, i.e., for inhibiting or disrupting oligomer formation ormaintenance. Thus, if one wishes to rapidly terminate the effect ofcellular activation, a monomeric ligand can be used. Conveniently, theparent ligand moiety can be modified at the same site as the multimer,using the same procedure, except substituting a monofunctional compoundfor the polyfunctional compound. Instead of the polyamines, monoamines,particularly of from 2 to 20 (although they can be longer), and usually2 to 12, carbon atoms can be used, such as ethylamine, hexylamine,benzylamine, etc. Alternatively, the monovalent parent compound can beused, in cases in which the parent compound does not have undueundesirable physiological activity (e.g. immunosuppression, mitogenesis,toxicity, etc.).

D. Illustrative hetero-oligomerizing (HED) and homo-oligomerizing (HOD)reagents with "bumps" that can bind to mutant receptors containingcompensatory mutations

As discussed above, one can prepare modified HED/HOD reagents that willfail to bind appreciably to their wildtype receptors (e.g., FKBP12) dueto the presence of substituents ("bumps") on the reagents thatsterically clash with sidechain residues in the receptor's bindingpocket. One may also make corresponding receptors that contain mutationsat the interfering residues ("compensatory mutations") and thereforegain the ability to bind ligands with bumps. Using "bumped" ligandmoieties and receptor domains bearing compensatory mutations shouldenhance the specificity and thus the potency of our reagents. Bumpedreagents should not bind to the endogenous, wildtype receptors, whichcan otherwise act as a "buffer" toward dimerizers based on naturalligand moieties. In addition, the generation of novel receptor-ligandpairs should simultaneously yield the HED reagents that will be usedwhen heterodimerization is required. For example, regulated vesiclefusion may be achieved by inducing the heterodimerization of syntaxin (aplasma membrane fusion protein) and synaptobrevin (a vesicle membranefusion protein) using a HED reagent This would not only provide aresearch tool, but could also serve as the basis of a gene therapytreatment for diabetes, using appropriately modified secretory cells.

As an illustration of "Bumped FK1012s" we prepared C10 acetamide andformamide derivatives of FK506. See FIG. 16A and our report, Spencer etal, "Controlling Signal Transduction with Synthetic Ligands," Science262 5136 (1993): 1019-1024 for additional details concerning thesyntheses of FK1012s A-C and FK506M. We chose to create two passes ofbumped FK1012: one with a bump at C10 and one at C9. The R- andS-isomers of the C10 acetamide and formamide of FK506 have beensynthesized according to the reaction sequence in FIG. 5B. These bumpedderivatives have lost at least three orders of magnitude in theirbinding affinity towards FKBP12 (FIG. 16A (panel B). The affinities weredetermined by measuring the ability of the derivatives to inhibitFKBP12's rotamase activity.

An illustrative member of a second class of C9-bumped derivatives is thespiro-epoxide (depicted in FIG. 16B (panel C), which has been preparedby adaptation of known procedures. See e.g. Fisher et al, J Org Chem 568(1991): 2900-7 and Edmunds et al, Tet Lett 32 48 (1991):819-820. Aparticularly interesting series of C9 derivatives are characterized bytheir sp3 hybridization and reduced oxidation state at C9. Several suchcompounds have been synthesized according to the reactions shown in FIG.16C.

It should be appreciated that heterodimers (and otherhetero-oligomerizers) must be constructed differently than thehomodimers, at least for applications where homodimer contaminationcould adversely affect their successful use. One illustrative syntheticstrategy developed to overcome this problem is outlined in FIG. 16B(panel D). Coupling of mono alloc-protected 1,6-hexanediamine (Stahl etal, J Org Chem 43 11 (1978): 2285-6) with a derivatized form of FK506 inmethylene chloride with an excess of triethylamine gave analloc-amine-substituted FK506 in 44% yield. This intermediate can now beused in the coupling with any activated FK506 (or bumped-FK506)molecule. Deprotection with catalytic tetrakis-triphenylphosphinepalladium in the presence of dimedone at rt in THF removes the amineprotecting group. Immediate treatment with an activated FK506derivative, followed by desilylation leads to a dimeric product. Thistechnique has been used to synthesize the illustrated HOD and HEDreagents.

E. Illustrative Cyclosporin-based reagents

Cyclosporin A (CsA) is a cyclic undecapeptide that binds with highaffinity (6 nM) to its intracellular receptor cyclophilin, an 18 kDamonomeric protein. The resulting complex, like the FPB12-FK506 complex,binds to and inactivates the protein phosphatase calcineurin resultingin the immunosuppressive properties of the drug. As a furtherillustration of this invention, we have dimerized CsA via its MeBmt1sidechain in 6 steps and 35% overall yield to give (CsA)2 (FIG. 17,steps 1-4 were conducted as reported in Eberle et al, J Org Chem 57 9(1992): 2689-91). As with FK1012, the site for dimerization was chosensuch that the resulting dimer can bind to two molecules of cydophilinyet cannot bind to calcineurin following cyclophilin-binding. We havedemonstrated that (CsA)2 binds to cyclophilin A with 1:2 stoichiometry.Hence, (CsA)2, like FK1012s, does not inhibit signaling pathways and isthus neither immunosuppressive nor toxic.

VIII. Target Gene

A. Transcription Initiation Region

The second construct or second series of constructs will have aresponsive element in the 5' region, which responds to ligand-mediatedoligomerization of the chimeric receptor protein, presumably via thegeneration and transduction of a transcription initiation signal asdiscussed infra. Therefore, it will be necessary to know at least onetranscription initiation system, e.g. factor, which is activated eitherdirectly or indirectly, by the cytoplasmic domain or can be activated byassociation of two domains. It will also be necessary to know at leastone promoter region which is responsive to the resulting transcriptioninitiation system. Either the promoter region or the gene under itstranscriptional control need be known. In other words, an action domaincan be selected for the chimeric proteins (encoded by a "first" seriesconstruct) based on the role of that action domain in initiatingtranscription via a given promoter or responsive element. See e.g.Section V(A) "Cytoplasmic domains", above.

Where the responsive element is known, it can be included in the targetgene construct to provide an expression cassette for integration intothe genome (whether episomally or by chromosomal incorporation). It isnot necessary to have isolated the particular sequence of the responsiveelement, so long as a gene is known which is transcriptionally activatedby the cytoplasmic domain upon natural ligand binding to the proteincomprising the cytoplasmic domain. Homologous recombination could thenbe used for insertion of the gene of interest downstream from thepromoter region to be under the transcriptional regulation of theendogenous promoter region. Where the specific responsive elementsequence is known, that can be used in conjunction with a differenttranscription initiation region, which can have other aspects, such as ahigh or low activity as to the rate of transcription, binding ofparticular transcription factors and the like.

The expression construct will therefore have at its 5' end in thedirection of transcription, the responsive element and the promotersequence which allows for induced transcription initiation of a targetgene of interest, usually a therapeutic gene. The transcriptionaltermination region is not as important, and can be used to enhance thelifetime of or make short half-lived mRNA by inserting AU sequenceswhich serve to reduce the stability of the mRNA and, therefore, limitthe period of action of the protein. Any region can be employed whichprovides for the necessary transcriptional termination, and asappropriate, translational termination.

The responsive element can be a single sequence or can be oligomerized,usually having not more than about 5 repeats, usually having about 3repeats.

Homologous recombination can also be used to remove or inactivateendogenous transcriptional control sequences, including promoter and/orresponsive elements, which are responsive to the oligomerization event,and/or to insert such responsive transcriptional control sequencesupstream of a desired endogenous gene.

B. Product

A wide variety of genes can be employed as the target gene, includinggenes that encode a protein of interest or an antisense sequence ofinterest or a ribozyme of interest. The target gene can be any sequenceof interest which provides a desired phenotype. The target gene canexpress a surface membrane protein, a secreted protein, a cytoplasmicprotein, or there can be a plurality of target genes which can expressdifferent types of products. The target gene may be an antisensesequence which can modulate a particular pathway by inhibiting atranscriptional regulation protein or turn on a particular pathway byinhibiting the translation of an inhibitor of the pathway. The targetgene can encode a ribozyme which may modulate a particular pathway byinterfering, at the RNA level, with the expression of a relevanttranscriptional regulator or with the expression of an inhibitor of aparticular pathway. The proteins which are expressed, singly or incombination, can involve homing, cytotoxicity, proliferation, immuneresponse, inflammatory response, clotting or dissolving of clots,hormonal regulation, or the like. The proteins expressed could benaturally-occurring, mutants of naturally-occurring proteins, uniquesequences, or combinations thereof.

The gene can be any gene which is secreted by a cell, so that theencoded product can be made available at will, whenever desired orneeded by the host. Various secreted products include hormones, such asinsulin, human growth hormone, glucagon, pituitary releasing factor,ACTH, melanotropin, relaxin, etc.; growth factors, such as EGF, IGF-1,TGF-α, -β, PDGF, G-CSF, M-CSF, GM-CSF, FGF, erythropoietin,megakaryocytic stimulating and growth factors, etc.; interleukins, suchas IL-1 to -13; TNFα and -β, etc.; and enzymes, such as tissueplasminogen activator, members of the complement cascade, perforins,superoxide dismutase, coagulation factors, antithrombin-III, FactorVIIIc, Factor VIIIvW, α-anti-trypsin, protein C, protein S, endorphins,dynorphin, bone morphogenetic protein, CFTR, etc.

The gene can be any gene which is naturally a surface membrane proteinor made so by introducing an appropriate signal peptide andtransmembrane sequence. Various proteins include homing receptors, e.g.L-selection (Mel-14), blood-related proteins, particularly having akringle structure, e.g. Factor VIIIc, Factor VIIIvW, hematopoietic cellmarkers, e.g. CD3, CD4, CD8, B cell receptor, TCR subunits α, β, γ, δ,CD10, CD19, CD28, CD33, CD38, CD41, etc., receptors, such as theinterleukin receptors IL-2R, IL-4R, etc., channel proteins, for influxor efflux of ions, eg. H⁺, Ca⁺², K⁺, Na⁺, Cl⁻, etc., and the like; CFTR,tyrosine activation motif, ζ activation protein, etc.

Proteins may be modified for transport to a vesicle for exocytosis. Byadding the sequence from a protein which is directed to vesicles, wherethe sequence is modified proximal to one or the other terminus, orsituated in an analogous position to the protein source, the modifiedprotein will be directed to the Golgi apparatus for packaging in avesicle. This process in conjunction with the presence of the chimericproteins for exocytosis allows for rapid transfer of the proteins to theextracellular medium and a relatively high localized concentration.

Also, intracellular proteins can be of interest, such as proteins inmetabolic pathways, regulatory proteins, steroid receptors,transcription factors, etc., particularly depending upon the nature ofthe host cell. Some of the proteins indicated above can also serve asintracellular proteins.

The following are a few illustrations of different genes. In T-cells,one may wish to introduce genes encoding one or both chains of a T-cellreceptor. For B-cells, one could provide the heavy and light chains foran immunoglobulin for secretion. For cutaneous cells, e.g.keratinocytes, particularly stem cells keratinocytes, one could providefor infectious protection, by secreting α-, β- or -γ interferon,antichemotactic factors, proteases specific for bacterial cell wallproteins, etc.

In addition to providing for expression of a gene having therapeuticvalue, there will be many situations where one may wish to direct a cellto a particular site. The site can include anatomical sites, such aslymph nodes, mucosal tissue, skin, synovium, lung or other internalorgans or functional sites, such as clots, injured sites, sites ofsurgical manipulation, inflammation, infection, etc. By providing forexpression of surface membrane proteins which will direct the host cellto the particular site by providing for binding at the host target siteto a naturally-occurring epitope, localized concentrations of a secretedproduct can be achieved. Proteins of interest include homing receptors,e.g. L-selectin, GMP140, CLAM-1, etc., or addressins, e.g. ELAM-1, PNAd,LNAd, etc., clot binding proteins, or cell surface proteins that respondto localized gradients of chemotactic factors. There are numeroussituations where one would wish to direct cells to a particular site,where release of a therapeutic product could be of great value.

In many situations one may wish to be able to kill the modified cells,where one wishes to terminate the treatment, the cells becomeneoplastic, in research where the absence of the cells after theirpresence is of interest, or other event. For this purpose one canprovide for the expression of the Fas antigen or TNF receptor fused to abinding domain. (Watanable-Fukunaga et al. Nature (1992) 356, 314-317).In the original modification, one can provide for constitutiveexpression of such constructs, so that the modified cells have suchproteins on their surface or present in their cytoplasm. Alternatively,one can provide for controlled expression, where the same or differentligand can initiate expression and initiate apoptosis. By providing forthe cytoplasmic portions of the Fas antigen or TNF receptor in thecytoplasm joined to binding regions different from the binding regionsassociated with expression of a target gene of interest, one can killthe modified cells under controlled conditions.

C. Illustrative Exemplifications

By way of illustration, cardiac patients or patients susceptible tostroke may be treated as follows. Cells modified as described herein maybe administered to the patient and retained for extended periods oftime. Illustrative cells include plasma cells, B-cells, T-cells, orother hematopoietic cells. The cell would be modified to express aprotein which binds to a blood clot, e.g. having a kringle domainstructure or an adhesive interactive protein, e.g. CD41, and to expressa dot dissolving protein, e.g. tissue plasminogen activator,streptokinase, etc. In this way, upon ligand-mediated oligomerization,the cells would accumulate at the site of the dot and provide for a highlocalized concentration of the thrombolytic protein.

Another example is reperfusion injury. Cells of limited lifetime couldbe employed, e.g. macrophages or polymorphonuclear leukocytes("neutrophils"). The cells would have a neutrophil homing receptor todirect the cells to a site of reperfusion injury. The cell would alsoexpress superoxide dismutase, to destroy singlet oxygen and inhibitradical attack on the tissue.

A third example is autoimmune disease. Cells of extended lifetime, e.g.T cells could be employed. The constructs would provide for a horningreceptor for homing to the site of autoimmune injury and for cytotoxicattack on cells causing the injury. The therapy would then be directedagainst cells causing the injury. Alternatively, one could provide forsecretion of soluble receptors or other peptide or protein, where thesecretion product would inhibit activation of the injury causing cellsor induce anergy. Another alternative would be to secrete anantiinflammatory product, which could serve to diminish the degenerativeeffects.

A fourth example involves treatment of chronic pain with endorphin viaencapsulation. A stock of human fibroblasts is transfected with aconstruct in which the chimeric transcriptional regulatory proteincontrols the transcription of human endorphin. The DNA constructconsists of three copies of the binding site for the HNF-1*transcription factor GTTAAGTTAAC [SEQ ID NO:2] upstream of a TATAAA siteand a transcriptional initiation site. The endorphin cDNA would beinserted downstream of the initiation site and upstream of apolyadenylation and termination sequences. Optionally, the endorphincDNA is outfitted with "PEST" sequences to make the protein unstable orAUUA sequences in the 3' nontranslated region of the mRNA to allow it tobe degraded quickly.

The fibroblasts are also transfected with a construct having twotranscription units, one of which would encode the HNF-1* cDNA truncatedto encode just the DNA binding sequences from amino adds 1 to 250coupled to a trimeric FKBP binding domain under the transcriptional andtranslational control of regulatory initiation and termination regionsfunctional in the fibroblasts. The construct would include an additionaltranscription unit driven by the same regulatory regions directing theproduction of a transcriptional activation domain derived from HNF-4coupled to trimeric FKBP'. (The prime intends an altered FKBP that bindsat nM concentration to a modified FK506. The modification inhibitsbinding to the endogenous FKBP.)

These genetically modified cells would be encapsulated to inhibit immunerecognition and placed under the patient's skin or other convenientinternal site. When the patient requires pain medication, the patientadministers a dimeric ligand FK506-FK506'where about 1 μg to 1 mg wouldsuffice. In this manner one could provide pain relief without injectionsor the danger of addiction.

A fifth example is the treatment of osteoporosis. Lymphocytes can beclonally developed or skin fibroblasts grown in culture from the patientto be treated. The cells would be transfected as described above, wherea bone morphogenic factor cDNA gene would replace the endorphin gene.For lymphocytes, antigen specific clones could be used which would allowtheir destruction with antibodies to the idiotype of the sIg. Inaddition, administration of the antigen for the sIg would expand thecell population to increase the amount of the protein which could bedelivered. The lymphocyte clones would be infused and the ligandadministered as required for production of the bone morphogenic factor.By monitoring the response to the ligand, one could adjust the amount ofbone morphogenic factor which is produced, so as to adjust the dosage tothe required level.

A sixth situation has general application in conjunction with genetherapies involving cells which may be required to be destroyed. Forexample, a modified cell may become cancerous or result in anotherpathologic state. Constructs would be transfected into the modifiedcells having the necessary transcriptional and translational regulatoryregions and encoding a protein which upon oligomerization results incell death, e.g. apoptosis. For example, the fas antigen or Apo-1antigen induces apoptosis in most cell tpes (Trauth et al. (1989)Science 245, 301-305; Watanaba-Fukunaga et al. (1992) Nature 356, 314).In this manner by co-transfecting the protective constructs into cellsused for gene therapy or other purpose, where there may be a need toensure the death of a portion or all of the cells, the cells may benotified to provide for controlled cytotoxicity by means of the ligand.

Another situation is to modify antigen specific T cells, where one canactivate expression of a protein product to activate the cells. The Tcell receptor could be directed against tumor cells, pathogens, cellsmediating autoimmunity, and the like. By providing for activation of thecells, for example, an interleulan such as IL-2, one could provide forexpansion of the modified T cells in response to a ligand. Other uses ofthe modified T cells would include expression of homing receptors fordirecting the T cells to specific sites, where cytotoxicity,upregulation of a surface membrane protein of target cells, e.g.endothelial cells, or other biological event would be desired.

Alternatively one may want to deliver high doses of cytotoxic factors tothe target site. For example, upon recognition of tumor antigens via ahoming receptor, tumor-infiltrating lymphocytes (TILs) may be triggeredto deliver toxic concentrations of TNF or other similar product.

Another alternative is to export hormones or factors which areexocytosed. By providing for enhanced exocytosis, a greater amount ofthe hormone or factor will be exported; in addition, if there is afeedback mechanism based on the amount of the hormone or factor in thecytoplasm, increased production of the hormone or factor will result.Or, one may provide for induced expression of the hormone or factor, sothat expression and export may be induced concomitantly.

One may also provide for proteins in retained body fluids, e.g. vascularsystem, lymph system, cerebrospinal fluid, etc. By modifying cells whichcan have an extended lifetime in the host, e.g. hematopoietic cells,keratinocytes, muscle cells, etc. particularly, stem cells, the proteinscan be maintained in the fluids for extended periods of time. The cellsmay be modified with constructs which provide for secretion orendocytosis. The constructs for secretion would have as thetranslocation domain, a signal peptide, and then as in the case of theother chimeric proteins, a binding domain and an action domain. Theaction domains may be derived from the same or different proteins. Forexample, with tissue plasminogen activator, one could have the clotbinding region as one action domain and the plasminogen active site as adifferent action domain. Alternatively, one could provide enhancedblockage of homing, by having a binding protein, such as LFA-1 as oneaction domain and a selection as a second action domain. By modifyingsubunits of proteins, e.g. integrins, T-cell receptor, sIg, or the like,one could provide soluble forms of surface membrane proteins which couldbe brought together to bind to a molecule. Other opportunities arecomplement proteins, platelet membrane proteins involved in clotting,autoantigens on the surface of cells, and pathogenic molecules on thesurface of infectious agents.

IX. Introduction of Constructs into Cells

The constructs can be introduced as one or more DNA molecules orconstructs, where there will usually be at least one marker and theremay be two or more markers, which will allow for selection of host cellswhich contain the construct(s). The constructs can be prepared inconventional ways, where the genes and regulatory regions may beisolated, as appropriate, ligated, cloned in an appropriate cloninghost, analyzed by restriction or sequencing, or other convenient means.Particularly, using PCR, individual fragments including all or portionsof a functional unit may be isolated, where one or more mutations may beintroduced using "primer repair", ligation, in vitro mutagensis, etc. asappropriate. The constructs once completed and demonstrated to have theappropriate sequences may then be introduced into the host cell by anyconvenient means. The constructs may be integrated and packaged intonon-replicating, defective viral genomes like Adenovirus,Adeno-associated virus (AAV), or Herpes simplex virus (HSV) or others,including retroviral vectors, for infection or transduction into cells.The constructs may include viral sequences for transfection, if desired.Alternatively, the construct may be introduced by fusion,electroporation, biolistics, transfection, lipofection, or the like. Thehost cells will usually be grown and expanded in culture beforeintroduction of the construct(s), followed by the appropriate treatmentfor introduction of the construct(s) and integration of theconstruct(s). The cells will then be expanded and screened by virtue ofa marker present in the construct. Various markers which may be usedsuccessfully include hprt, neomycin resistance, thymidine kinase,hygromycin resistance, etc.

In some instances, one may have a target site for homologousrecombination, where it is desired that a construct be integrated at aparticular locus. For example, can knock-out an endogenous gene andreplace it (at the same locus or elswhere) with the gene encoded for bythe construct using materials and methods as are known in the art forhomologous recombination. Alternatively, instead of providing a gene,one may modify the transcriptional initiation region of an endogenousgene to be responsive to the signal initiating domain. aIn suchembodiments, transcription of an endogenous gene such as EPO, tPA, SOD,or the like, would be controlled by administration of the ligand. Forhomologous recombination, one may use either Ω or ◯-vectors. See, forexample, Thomas and Capecchi, Cell (1987) 51, 503-512; Mansour, et al.,Nature (1988) 336, 348-352; and Joyner, et al., Nature (1989) 338,153-156.

The constructs maybe introduced as a single DNA molecule encoding all ofthe genes, or different DNA molecules having one or more genes. Theconstructs may be introduced simultaneously or consecutively, each withthe same or different markers. In an illustrative example, one constructwould contain a therapeutic gene under the control of a specificresponsive element (e.g. NFAT), another encoding the receptor fusionprotein comprising the signaling region fused to the ligand receptordomain (e.g. as in MZF3E). A third DNA molecule encoding a homingreceptor or other product that increases the efficiency of delivery ofthe therapeutic product may also be introduced.

Vectors containing useful elements such as bacterial or yeast origins ofreplication, selectable and/or amplifiable markers, promoter/enhancerelements for expression in procaryotes or eucaryotes, etc. which may beused to prepare stocks of construct DNAs and for carrying outtransfections are well known in the art, and many are commerciallyavailable.

X. Administration of Cells and Ligands

The cells which have been modified with the DNA constructs are thengrown in culture under selective conditions and cells which are selectedas having the construct may then be expanded and further analyzed,using, for example, the polymerase chain reaction for determining thepresence of the construct in the host cells. Once the modified hostcells have been identified, they may then be used as planned, e.g. grownin culture or introduced into a host organism.

Depending upon the nature of the cells, the cells may be introduced intoa host organism, e.g. a mammal, in a wide variety of ways. Hematopoieticcells may be administered by injection into the vascular system, therebeing usually at least about 10⁴ cells and generally not more than about10¹⁰, more usually not more than about 10⁸ cells. The number of cellswhich are employed will depend upon a number of circumstances, thepurpose for the introduction, the lifetime of the cells, the protocol tobe used, for example, the number of administrations, the ability of thecells to multiply, the stability of the therapeutic agent, thephysiologic need for the therapeutic agent, and the like. Alternatively,with skin cells which may be used as a graft, the number of cells woulddepend upon the size of the layer to be applied to the burn or otherlesion. Generally, for myoblasts or fibroblasts, the number of cellswill at least about 104 and not more than about 10⁸ and may be appliedas a dispersion, generally being injected at or near the site ofinterest. The cells will usually be in a physiologically-acceptablemedium.

Instead of ex vivo modification of the cells, in many situations one maywish to modify cells in vivo. For this purpose, various techniques havebeen developed for modification of target tissue and cells in vivo. Anumber of virus vectors have been developed, such as adenovirus andretroviruses, which allow for transfection and random integration of thevirus into the host See, for example, Dubensky et al. (1984) Proc. Natl.Acad. Sci. USA 81, 7529-7533; Kaneda et al., (1989) Science 243,375-378;Hiebert et al. (1989) Proc. Natl. Acad. Sci. USA 86, 3594-3598; Hatzogluet al. (1990) J. Biol. Chem. 265, 17285-17293 and Ferry, et al. (1991)Proc. Natl. Acad. Sci. USA 88, 8377-8381. The vector may be administeredby injection, e.g. intravascularly or intramuscularly, inhalation, orother parenteral mode.

In accordance with in vivo genetic modification, the manner of themodification will depend on the nature of the tissue, the efficiency ofcellular modification required, the number of opportunities to modifythe particular cells, the accessibility of the tissue to the DNAcomposition to be introduced, and the like. By employing an attenuatedor modified retrovirus carrying a target transcriptional initiationregion, if desired, one can activate the virus using one of the subjecttranscription factor constructs, so that the virus may be produced andtransfect adjacent cells.

The DNA introduction need not result in integration in every case. Insome situations, transient maintenance of the DNA introduced may besufficient. In this way, one could have a short term effect, where cellscould be introduced into the host and then turned on after apredetermined time, for example, after the cells have been able to hometo a particular site.

The ligand providing for activation of the cytoplasmic domain may thenbe administered as desired. Depending upon the binding affinity of theligand, the response desired, the manner of administration, thehalf-life, the number of cells present, various protocols may beemployed. The ligand may be administered parenterally or orally. Thenumber of administrations will depend upon the factors described above.The ligand may be taken orally as a pill, powder, or dispersion;bucally; sublingually; injected intravascularly, intraperitoneally,subcutaneously; by inhalation, or the like. The ligand (and monomericcompound) may be formulated using conventional methods and materialswell known in the art for the various routes of administration. Theprecise dose and particular method of administration will depend uponthe above factors and be determined by the attending physician or humanor animal healthcare provider. For the most part, the manner ofadministration will be determined empirically.

In the event that the activation by the ligand is to be reversed, themonomeric compound may be administered or other single binding sitecompound which can compete with the ligand. Thus, in the case of anadverse reaction or the desire to terminate the therapeutic effect, themonomeric binding compound can be administered in any convenient way,particularly intravascularly, if a rapid reversal is desired.Alternatively, one may provide for the presence of an inactivationdomain with a DNA binding domain, or apoptosis by having Fas or TNFreceptor present as constitutively expressed constructs.

The particular dosage of the ligand for any application may bedetermined in accordance with the procedures used for therapeutic dosagemonitoring, where maintenance of a particular level of expression isdesired over an extended period of times, for example, greater thanabout two weeks, or where there is repetitive therapy, with individualor repeated doses of ligand over short periods of time, with extendedintervals, for example, two weeks or more. A dose of the ligand within apredetermined range would be given and monitored for response, so as toobtain a time-expression level relationship, as well as observingtherapeutic response. Depending on the levels observed during the timeperiod and the therapeutic response, one could provide a larger orsmaller dose the next time, following the response. This process wouldbe iteratively repeated until one obtained a dosage within thetherapeutic range. Where the ligand is chronically administered, oncethe maintenance dosage of the ligand is determined, one could then doassays at extended intervals to be assured that the cellular system isproviding the appropriate response and level of the expression product.

It should be appreciated that the system is subject to many variables,such as the cellular response to the ligand, the efficiency ofexpression and, as appropriate, the level of secretion, the activity ofthe expression product, the particular need of the patient, which mayvary with time and circumstances, the rate of loss of the cellularactivity as a result of loss of cells or expression activity ofindividual cells, and the like. Therefore, it is expected that for eachindividual patient, even if there were universal cells which could beadministered to the population at large, each patient would be monitoredfor the proper dosage for the individual.

The subject methodology and compositions may be used for the treatmentof a wide variety of conditions and indications. For example, B- andT-cells may be used in the treatment of cancer, infectious diseases,metabolic deficiencies, cardiovascular disease, hereditary coagulationdeficiencies, autoimmune diseases, joint degenerative diseases, e.g.arthritis, pulmonary disease, kidney disease, endocrine abnormalities,etc. Various cells involved with structure, such as fibroblasts andmyoblasts, may be used in the treatment of genetic deficiencies, such asconnective tissue deficiencies, arthritis, hepatic disease, etc.Hepatocytes could be used in cases where large amounts of a protein mustbe made to complement a deficiency or to deliver a therapeutic productto the liver or portal circulation.

The following examples are offered by way illustration and not by waylimitation.

EXAMPLES

Cellular Transformations and Evaluation

Example 1: Induction of Isolated IL-2 Enhancer-Binding TranscriptionFactors by Cross-Linking the CD3 Chain of the T-Cell Receptor.

The plasmid pSXNeo/IL2 (IL2-SX) (FIG. 1), which contains the placentalsecreted alkaline phosphatase gene under the control of human IL-2promoter (-325 to +47; MCB(86) 6, 3042), and related plasmid variants(i.e. NFAT-SX, NF B-SX, OAP/Oct1-SX, and AP-1-SX) in which the reportergene is under the transcriptional control of the minimal IL-2 promoter(-325 to -294 and -72 to +47) combined with synthetic oligomerscontaining various promoter elements (i.e. NFAT, NK B, OAP/Oct-1, andAP1, respectively), were made by three piece ligations of 1) pPL/SEAP(Berger, et al., Gene (1988) 66,1) cut with SspI and HindIII; 2)pSV2/Neo (Southern and Berg, J. Mol. Appl. Genet. (1982) 1, 332) cutwith NdeI, blunted with Klenow, then cut with PvuI; and 3) variouspromoter-containing plasmids (i.e. NFAT-CD8, B-8, cx12lacZ-Oct-1,AP1-LUCIF3H, or cx15IL2) (described below) cut with PvuI and HindIII.NFAT-CD8 contains 3 copies of the NFAT-binding site (-286 to -257; Genesand Dev. (1990) 4, 1823) and cx12lacZ-Oct contains 4 copies of theOAP/Oct-1/(ARRE-1) binding site (MCB, (1988) 8,1715) from the human IL-2enhancer; B-CD8 contains 3 copies of the NF B binding site from themurine light chain (EMBO (1990) 9, 4425) and AP1-LUCIF3H contains 5copies of the AP-1 site (5'-TGA- CTCAGCGC-3'[SEQ ID NO:3]) from themetallothionen promoter.

In each transfection, 5 μg of expression vector, pCDL-SR (MCB 8,466-72)(Tac-IL2 receptor -chain), encoding the chimeric receptor TAC/TAC/Z(TTZ) (PNAS 88, 8905-8909), was co-transfected along with varioussecreted alkaline phosphatase-based reporter plasmids (see map ofpSXNeo/IL2 in FIG. 1) in TAg Jurkat cells (a derivative of the humanT-cell leukemia line Jurkat stably transfected with the SV40 large Tantigen (Northrup, et al., J. Biol. Chem. [1993]). Each reporter plasmidcontains a multimerized oligonudeotide of the binding site for adistinct IL-2 enhancer-binding transcription factor within the contextof the minimal IL-2 promoter or, alternatively, the intact IL-2enhancer/promoter upstream of the reporter gene. After 24 hours,aliquots of cells (approximately 10⁵) were placed in microtiter wellscontaining log dilutions of bound anti-TAC (CD25) mAb (33B3.1; AMAC,Westbrook, Me.) As a positive control and to control for transfectionefficiency, ionomycin (1 μm) and PMA (25 ng/ml) were added to aliquotsfrom each transfection. After an additional 14 hour incubation, thesupernatants were assayed for the alkaline phosphatase activity andthese activities were expressed relative to that of the positive controlsamples. The addition of 1 ng/ml FK506 dropped all activity due to NFATto background levels, demonstrating that deactivations are in the samepathway as that blocked by FK506. Each data point obtained was theaverage of two samples and the experiment was performed several timeswith similar results. See FIG. 5. The data show that with a knownextracellular receptor, one obtains an appropriate response with areporter gene and different enhancers. Similar results were obtainedwhen a MAb against the TcR complex (i.e. OKT3) was employed.

Example 2: Inhibitory Activity of the Immunosuppressant Drugs FK506 andCyclosporin A (CsA) or the Dimeric Derivative Compounds FK1012A (8),FK1012B (5), and CsA dimer (PB-1-218).

Ionomycin (1 μm) and PMA (25 ng/ml) were added to 10⁵ TAg-Jurkat cells.In addition, titrations of the various drugs were added. After 5 hoursthe cells were lysed in mild detergent (i.e. Triton X-100) and theextracts were incubated with the β-galactosidase substrate, MUG (methylgalactosidyl umbelliferone) for 1 hour. A glycine/EDTA stop buffer wasadded and the extracts assayed for fluorescence. Each data pointobtained was the average of two samples and the experiment was performedseveral times with similar results. Curiously, FK1012B appears toaugment mitogen activity slightly at the highest concentration (i.e. 5μg/ml); however, a control experiment shows that FK1012B is notstimulatory by itself. See FIG. 6.

Example 3. Activity of the Dimeric FK506 Derivative, FK1012A, on theChimeric FKBP12/CD3 (1FK3) Receptor.

5 μg of the eukaryotic expression vector, pBJ5, (based on pCDL-SR with apolylinker inserted between the 16 S splice site and the poly A site),containing the chimeric receptor (1FK3), was co-transfected with 4 μg ofthe NFAT-inducible secreted alkaline phosphatase reporter plasmid,NFAT-SX. As a control, 5 μg of pBJ5 was used, instead of 1FK3/pBJ5, in aparallel transfection. After 24 hours, aliquots of each transfectioncontaining approximately 10⁵ cells were incubated with log dilutions ofthe drug, FK1012A, as indicated. As a positive control and to controlfor transfection efficiency, ionomycin (1 μm) and PMA (25 ng/ml) wereadded to aliquots from each transfection. After an additional 14 hourincubation, the supernatants were assayed for alkaline phosphataseactivity and these activities were expressed relative to that of thepositive control samples. The addition of 2 ng/ml FK506 dropped allstimulations to background levels, demonstrating that the activationsare in the same pathway as that blocked by FK506. Hence, FK506 orcyclosporin will serve as effective antidotes to the use of thesecompounds. Each data point obtained was the average of two samples andthe experiment was performed several times with similar results. SeeFIG. 7.

Example 4A. Activity of the Dimeric FK506 Derivative, FK1012B, on theMyristoylated Chimeric CD3/FKBP12 (MZF3E) Receptor.

We have successfully demonstrated a number of approaches to liganddesign and syntheses, including positive results with FK506-based HODreagents named "FK1012"s. We have found that FK1012s achieve highaffinity, 2:1 binding stoichiometry (K_(d) (1)=0.1 nM; K_(d) (2)=0.8 nM)and do not inhibit calcineurin-mediated TCR signaling. The ligands areneither "immunosuppressive" nor toxic (up to 0.1 mM in cell culture).Similarly, we have prepared a cyclosporin A-based homodimerizing agent,"(CsA)2" which binds to the CsA receptor, cyclophylin, with 1:2stoichiometry, but which does not bind to calcineurin. Thus, likeFK1012s, (CsA)2 does not inhibit signalling pathways and is thus neitherimmunosuppressive nor toxic.

These and other of our examples of ligand-mediated protein associationresulted in the control of a signal transduction pathway. In anillustrative case, this was accomplished by creating an intracellularreceptor comprised of a small fragment of Src sufficient forposttranslational myristoylation (M), the cytoplasmic tail of zeta (Z; acomponent of the B cell receptor was also used), three consecutiveFKBP12s (F3) and a flu epitope tag (E). Upon expressing the constructMZF3E (FIG. 18) in human (Jurkat) T cells, we confirmed that the encodedchimeric protein underwent FK1012-mediated oligomerization. Theattendant aggregation of the zeta chains led to signaling via theendogenous TCR-signaling pathway (FIG. 15), as evidenced by secretion ofalkaline phosphatase (SEAP) in response to an FK1012 (EC₅₀ =50 nM). Thepromoter of the SEAP reporter gene was constructed to betranscriptionally activated by nuclear factor of activated T cells(NFAT), which is assembled in the nucleus following TCR-signaling.FK1012-induced signaling can be terminated by a deaggregation processinduced by a nontoxic, monomeric version of the ligand called FK506-M.

Specifically, 5 μg of the eukaryotic expression vector, pBJ5, containinga myristoylated chimeric receptor was co-transfected with 4 μg NFAT-SXMZE, MZF1E, MZF2E and MZF3E contain 0, 1, 2, or 3 copies of FKBP12,respectively, downstream of a myristoylated CD3 cytoplasmic domain (seeFIG. 2). As a control, 5 μg of pBJ5 was used in a parallel transfection.After 24 hours, aliquots of each transfection containing approximately10⁵ cells were incubated with log dilutions of the drug, FK1012B, asindicated. As a positive control and to control for transfectionefficiency, ionomycin (1 μm) and PMA (25 ng/ml) were added to aliquotsfrom each transfection. After an additional 12 hour incubation, thesupernatants were assayed for alkaline phosphatase activity and theseactivities were expressed relative to that of the positive controlsamples. The addition of 1 ng/ml FK506 dropped all stimulations to nearbackground levels, demonstrating that the activations are in the samepathway as that blocked by FK506. This result is further evidence of thereversibility of the subject cell activation. Each data point obtainedwas the average of two samples and the experiment was performed severaltimes with similar results. See FIG. 8. The myristoylated derivativesrespond to lower concentrations of the ligand by about an order ofmagnitude and activate NF-AT dependent transcription to comparablelevels, but it should be noted that the ligands are different. CompareFIGS. 7 and 8.

In vivo FK1012-induced protein dimerization We next wanted to confirmthat intracellular aggregation of the MZF3E receptor is indeed inducedby the FK1012. The influenza haemagglutinin epitope-tag (flu) of theMZF3E-construct was therefore exchanged with a different epitope-tag(flag-M2). The closely related chimeras, MZF3E_(flu) and MZF3E_(flag),were coexpressed in Jurkat T cells. Immunoprecipitation experimentsusing anti-Flag-antibodies coupled to agarose beads were performed afterthe cells were treated with FK1012A. In the presence of FK1012 (1 μM)the protein chimera MZF3E_(flag) interacts with MZF3E_(flu) and iscoimmunoprecipitated with MZF3E_(flag). In absence of FK1012A, nocoimmunoprecipitation of MZF3E_(flu) is observed. Related experimentswith FKBP monomer constructs MZF1E_(flu) and MZF1E_(flag), which do notsignal, revealed that they are also dimerized by FK1012A (FIG. 19). Thisreflects the requirement for aggregation observed with both theendogenous T cell receptor and our artificial receptor MZF3E.

FK1012-induced protein-tyrosine phosphorylation. The intracellulardomains of the TCR, CD3 and zeta-chains interact with cytoplasmicprotein tyrosine kinases following antigen stimulation. Specific membersof the Src family (Ick and/or fyn) phosphorylate one or more tyrosineresidues of activation motifs within these intracellular domains(tyrosine activation motif, TAM). The tyrosine kinase ZAP-70 isrecruited (via its two SH2 domains) to the tyrosine phosphorylatedT-cell-receptor, activated, and is likely to be involved in the furtherdownstream activation of phospholipase C. Addition of either anti-CD3MAb or FK1012A to Jurkat cells stably transfected with MZF3E resulted inthe recruitment of kinase activity to the zeta-chain as measured by anin vitro kinase assay following immunoprecipitation of the endogenous Tcell receptor zeta chain and the MZF3E-construct, respectively. Tyrosinephosphorylation after treatment of cells with either anti-CD3 MAb orFK1012 was detected using monoclonal alpha-phosphotyrosine antibodies.Whole cell lysates were analysed at varying times after stimulation. Asimilar pattern of tyrosine-phosphorylated proteins was observed afterstimulation with either anti-CD3 MAb or FK1012. The pattern consisted ofa major band of 70 kDa, probably ZAP-70, and minor bands of 120 kDa, 62kDa, 55 kDa and 42 kDa.

Example 4(B): Regulation of Programmed Cell Death with Immunophilin-FasAntigen Chimeras

The Fas antigen is a member of the nerve growth factor (NGF)/tumornecrosis factor (TNF) receptor superfamily of cell surface receptors.Crosslinking of the Fas antigen with antibodies to its extracellulardomain activates a poorly understood signaling pathway that results inprogrammed cell death or apoptosis. The Fas antigen and its associatedapoptotic signaling pathway are present in most cells including possiblyall tumor cells. The pathway leads to a rapid and unique cell death (2h) that is characterized by condensed cytoplasm, the absence of aninflammatory response and fragmentation of nucleosomal DNA, none ofwhich are seen in necrotic cell death.

We have also developed a second, inducible signaling system that leadsto apoptotic cell death. Like the MZF3E pathway, this one is initiatedby activating an artificial receptor that is the product of aconstitutively expressed "responder" gene. However, the new pathwaydiffers from the first in that our HOD reagents induce the synthesis ofproducts of an endogenous pathway rather than of the product of atransfected, inducible (e.g., reporter) gene.

Gaining control over the Fas pathway could have important implicationsfor biological research and medicine in the future. Transgenic animalsmight be created with "death" responder genes under the control ofcell-specific promoters. Target cells could then be chemically ablatedin the adult animal by treating it with a HOD reagent. In this way, therole of specific brain cells in memory or cognition or immune cells inthe induction and maintenance of autoimmune disorders could be assessed.Death responder genes might be introduced into tumors using the humangene therapy technique developed by M. Blaese and co-workers (Culver etal, Science 256 5063 (1992): 1550-2) and then subsequently activated bytreating the patient with a HOD reagent (in analogy to the "gancyclovir"gene therapy clinical trials recently reported for the treatment ofbrain tumors). Finally, we contemplate a component of gene therapy inthe future that would involve the coadministration of a death-respondergene together with the therapeutic gene. This would provide a "failsafe"component to gene therapy. If something were to go awry (a commonlydiscussed concern is an integration-induced loss of a tumor suppressorgene leading to cancer), the gene therapy patient could take a"failsafe" pill that would kill all transfected cells. This conceptcaused us to focus on the development of an orthogonal system of HODreagents. Thus, we desired a second set of reagents that have nopossibility of cross-reacting with the first, which would be used toturn on or off the transcription of therapeutic genes.

A chimeric cDNA has been constructed consisting of three FKBP12 domainsfused to the cytoplasmic signaling domain of the Fas antigen (FIG. 20).This construct, when expressed in human Jurkat and murine D10 T cells,can be induced to dimerize by an FK1012 reagent and initiate a signalingcascade resulting in FK1012-dependent apoptosis. The LD₅₀ forFK1012A-mediated death of cells transiently transfected with MFF3E is 15nM as determined by a loss of reporter gene activity (FIG. 20; for adiscussion of the assay, see legend to FIG. 21). These data coincidewith measurements of cell death in stably transfected cell lines. Sincethe stable transfectants represent a homogeneous population of cells,they have been used to ascertain that death is due to apoptosis ratherthan necrosis (membrane blebbing, nudeosomal DNA fragmentation).However, the transient transfection protocol requires much less work andhas therefore been used as an initial assay system, as described below.

Example 4(C): Regulation of Programmed Cell Death with Cyclophilin-FasAntigen Chimeras

We have also prepared a series of cyclophilin C-Fas antigen constructsand assayed their ability to induce (CsA)2-dependent apoptosis intransient expression assays (FIG. 21A). In addition, (CsA)2-dependentapoptosis has been demonstrated with human Jurkat T cells stablytransfected with the most active construct in the series, MC3FE(M=myristoylation domain of Src, C=cyclophilin domain, F=cytoplasmictail of Fas, E=flu epitope tag). The cytoplasmic tail of Fas was fusedeither before of after 1, 2,3, or 4 consecutive cyclophilin domains. Twocontrol constructs were also prepared that lack the Fas domain. In thiscase we observed that the signaling domain functions only when placedafter the dimerization domains. (The zeta chain constructs signal whenplaced either before or after the dimerization domains.) Both theexpression levels of the eight signaling constructs, as ascertained byWestern blotting, and their activities differed quantitatively (FIG.21B). The optimal system has thus far proved to be MC3FE. The LD₅₀ for(CsA)2-mediated cell death with MC3FE is ˜200 nM. These data demonstratethe utility of the cydophilin-cyclosporin interactions for regulatingintracellular protein association and illustrate an orthogonal reagentsystem that will not cross-react with the FKBP12-FK1012 system. Further,in this case, the data show that only dimerization and not aggregationis required for initiation of signal transduction by the Fas cytoplasmictail.

Mutation of the N-terminal glycine of the myristoylation signal to analanine prevents myristoylation and hence membrane localization. We havealso observed that the mutated construct (ΔMFF3E) was equally potent asan inducer of FK1012-dependent apoptosis, indicating that membranelocalization is not necessary for Fas-mediated cell death.

Example 5. Construction of Murine Signalling Chimeric Protein.

The various fragments were obtained by using primers described in FIG.4. In referring to primer numbers, reference should be made to FIG. 4.

An approximately 1.2 kb cDNA fragment comprising the I-E chain of themurine class II MHC receptor (Cell, 32,745) was used as a source of thesignal peptide, employing P#6048 [SEQ ID NO:4]and P#6049 [SEQ ID NO:6]togive a 70 bp SacII-XhoI fragment using PCR as described by the supplier(Promega). A second fragment was obtained using a plasmid comprising Tac(IL2 receptor chain) joined to the transmembrane and cytoplasmic domainsof CD3 (PNAS, 88,8905). Using P#6050 [SEQ ID NO:8]and P#6051, [SEQ IDNO:10]a 320 bp XhoI-EcoRI fragment was obtained by PCR comprising thetransmembrane and cytoplasmic domains of CD3. These two fragments wereligated and inserted into a SacII-EcoRI digested pBluescript(Stratagene) to provide plasmid, SPZ/KS.

To obtain the binding domain for FK506, plasmid rhFKBP (provided by S.Schreiber, Nature (1990) 346, 674) was used with P#6052 [SEQ IDNO:33]and P#6053 [SEQ ID NO:35]to obtain a 340 bp XhoI-SalI fragmentcontaining human FKBP12. This fragment was inserted into pBluescriptdigested with XhoI and SalI to provide plasmid FK12/KS, which was thesource for the FKBP12 binding domain. SPZ/KS was digested with XhoI,phosphatased (cell intestinal alkaline phosphatase; CIP) to preventself-annealing, and combined with a 10-fold molar excess of theXhoI-SalI FKBP12-containing fragment from FK12/KS. Clones were isolatedthat contained monomers, dimers, and trimers of FKBP12 in the correctorientation. The clones 1FK1/KS, 1FK2/KS, and 1FK3/KS are comprised ofin the direction of transcription; the signal peptide from the murineMHC class II gene I-E , a monomer, dimer or trimer, respectively, ofhuman FKBP12, and the transmembrane and cytoplasmic portions of CD3.Lastly, the SacII-EcoRI fragments were excised from pBluescript usingrestriction enzymes and ligated into the polylinker of pBJ5 digestedwith SacII and EcoRI to create plasmids 1FK1/pBJ5, 1FK2/pBJ5, and1FK3/pBJ5, respectively. See FIGS. 3 and 4.

Example 6

A. Construction of Intracellular Signaling Chimera.

A myristoylation sequence from c-src was obtained from Pellman, et al.,Nature 314,374, and joined to a complementary sequence of CD3 to providea primer which was complementary to a sequence 3' of the transmembranedomain, namely P#8908 [SEQ ID NO:23]. This primer has a SacII siteadjacent to the 5' terminus and a XhoI sequence adjacent to the 3'terminus of the myristoylation sequence. The other primer P#8462 [SEQ IDNO:12]has a SalI recognition site 3' of the sequence complementary tothe 3' terminus of CD3, a stop codon and an EcoRI recognition site.Using PCR, a 450 bp SacII-EcoRI fragment was obtained, which wascomprised of the myristoylation sequence and the CD3 sequence fused inthe 5' to 3' direction. This fragment was ligated intoSacII/EcoRI-digested pBJ5 (XhoI)(SalI and cloned, resulting in plasmidMZ/pBJ5. Lastly, MZ/pBJ5 was digested with SalI, phosphatased, andcombined with a 10-fold molar excess of the XhoI-SalI FKBP12-containingfragment from FK12/KS and ligated. After cloning, the plasmidscomprising the desired constructs having the myristoylation sequence,CD3 and FKBP12 multimers in the 5'-3' direction were isolated andverified as having the correct structure. See FIGS. 2 and 4.

B. Construction of expression cassettes for intracellular signalingchimeras

The construct MZ/pBJ5 (MZE/pBJ5) is digested with restriction enzymesXhoI and SalI, the TCR ζ fragment is removed and the resulting vector isligated with a 10 fold excess of a monomer, dimer, trimer or higherorder multimer of FKBP12 to make MF1E, MF2E, MF3E or MF_(n) E/pBJ5.Active domains designed to contain compatible flanking restriction sites(i.e. XhoI and SalI) can then be cloned into the unique XhoI or SalIrestriction sites of MF_(n) E/pBJ5.

Example 7. Construction of Nuclear Chimera

A. GAL4 DNA binding domain--FKBP domain(s)--epitope tag. The GAL4 DNAbinding domain (amino acids 1-147) was amplified by PCR using a 5'primer (#37) that contains a SaclI site upstream of a Kozak sequence anda translational start site, and a 3' primer (#38) that contains a SalIsite. The PCR product was isolated, digested with SacII and SaII, andligated into pBluescript II KS (+) at the Sacll and Sall Sites,generating the construct pBS-GAL4. The construct was verified bysequencing. The SacII/SaII fragment from pBS-GAL4 was isolated andligated into the IFK1/pBJ5 and IFK3/pBJ5 constructs (containing themyristoylation sequence, see Example 6) at the SacII and Xhol sites,generating constructs GF1E, GF2E and GF3E.

5' end of PCR amplified product:

           SacII          |----Gal4(1-147)--->>                                                   M  K  L  L  S  S  I [SEQ ID NO:44]                     5'   CGACACCGCGGCCACCATGAAGCTACTGTCTTCTATCG[SEQ ID NO:41]                                    Kozak                                                          3' end of PCR amplified product:                                               -       <<----Gal4(1-147----)|                                           R  Q  L  T  V  S [SEQ ID NO:46]                                          5'   GACAGTTGACTGTATCGGTCGACTGTCG [SEQ ID NO:45]                              3'   CTGTCAACTGACATAGCCAGCTGACAGC [SEQ ID NO:77]                                                SalI                                                  

B. HNF1 dimerization/DNA binding domain--FKBP domain(s)--tag. The HNF1adimerization/DNA binding domain (amino acids 1-282) was amplified by PCRusing a 5' primer (#39) that contains a SacII site upstream of a Kozaksequence and a translational start site, and a 3' primer (#40) thatcontains a SalI site. The PCR product was isolated, digested with SacIIand SalI, and ligated into pBluescript II KS (+) at the SacII and SalIsites, generating the construct pBS-HNF. The construct was verified bysequencing. The SacII/SalI fragment from pBS-HNF was isolated andligated into the IFK1/pBJ5 and IFK3/pBJ5 constructs at the SacII andXhoI sites, generating constructs HF1E, HF2E and HF3E.

5' end of PCR amplified product:

    SacII              |--HNF1(1-281)-->>                                                       M  V  S  K  L  S [SEQ ID NO:50]                          5'   CGACACCGCGGCCACCATGGTTTCTAAGCTGAGC [SEQ ID NO:49]                                   Kozak                                                               - 3' end of PCR amplified product:                                                <<----HNF1(1-282)--|                                                  A  F  R  H  K  L [SEQ ID NO:52]                                         5'   CCTTCCGGCACAAGTTGGTCGACTGTCG [SEQ ID NO:51]                              3'   GGAAGGCCGTGTTCAACCAGCTGACAGC [SEQ ID NO:78]                                                SalI                                                  

C. FKBP domain(s)-VP16 transcrip. activation domain(s)-epitope tag.

These constructs were made in three steps: (i) a construct was createdfrom IPK3/pBJ5 in which the myristoylation sequence was replaced by astart site immediately upstream of an XhoI site, generating constructSF3E; (ii) a nuclear localization sequence was inserted into the XhoIsite, generating construct NF3E; (iii) the VP16 activation domain wascloned into the SalI site of NF3E, generating construct NF3V1E.

(i). Complementary oligonucleotides (#45 and #46) encoding a Kozaksequence and start site flanked by SacII and XhoI sites were annealed,phosphorylated and ligated into the SacII and XhoI site of MF3E,generating construct SF3E.

Insertion of generic start site

               Kozak                                                                               M  L  E [SEQ ID NO:54]                                         5'     GGCCACCATGC [SEQ ID NO:53]                                             3'   CGCCGGTGGTACGAGCT [SEQ ID NO:79]                                              SacII        XhoI                                                             overhang     overhang                                              

(ii). Complementary oligonucleotides (#47 and #48) encoding the SV40 Tantigen nuclear localization sequence flanked by a 5' SalI site and a 3'XhoI site were annealed, phosphorylated and ligated into the XhoI siteof SF1E, generating the construct NF1E. The construct was verified byDNA sequencing. A construct containing the mutant or defective form ofthe nuclear localization sequence, in which a threonine is substitutedfor the lysine at position 128, was also isolated. This is designatedNF1E-M. Multimers of the FKBP12 domain were obtained by isolating theFKBP12 sequence as an XhoI/SalI fragment from pBS-FKBP12 and ligatingthis fragment into NF1E linearized with XhoI. This resulted in thegeneration of the constructs NF2E and NF3E.

Insertion of NLS into generic start site

                  T (ACN)                                                                  126               132                                                   L  D  P  K  K  K  R  K  V  L  E [SEQ ID NO:59]                               5' TCGACCCTAAGAAGAAGAGAAAGGTAC [SEQ ID NO:58]                                 3'     GGGATTCTTCTTCTCTTTCCATGAGCT [SEQ ID NO:80]                                 SalI                      XhoI                                      

Threonine at position 128 results in a defective NLS.

(iii). The VP16 transcriptional activation domain (amino adds 413-490)was amplified by PCR using a 5' primer (#43) that contains SalI site anda 3' primer (#44) that contains an XhoI site. The PCR product wasisolated, digested with SalI and XhoI, and ligated into MF3E at the XhoIand SalI sites, generating the construct MV1E. The construct wasverified by sequencing. Multimerized VP16 domains were created byisolating the single VP16 sequence as a XhoI/SalI fragment from MV1E andligating this fragment into MV1E linearized with XhoI. Constructs MV2E,MV3E and MV4E were generated in this manner. DNA fragments encoding oneor more multiple VP16 domains were isolated as XhoI/SalI fragments fromMV1E or MV2E and ligated into NF1E linearized with SalI, generating theconstructs NF1V1E and NF1V3E. Multimers of the FKBP12 domain wereobtained by isolating the FKBP12 sequence as an XhoI/SalI fragment frompBS-FKBP12 and ligating this fragment into NF1V1E linearized with XhoI.This resulted in the generation of the constructs NF2V1E and NF3V1E.

5' end of PCR amplified product:

              SalI   |--VP16(413-490)--->>                                                   A  P  P  T  D  V [SEQ ID NO:64]                             5'    CGACAGTCGACGCCCCCCCGACCGATGTC [SEQ ID NO:61]                            3' end of PCR amplified product:                                               <<-- VP16(413-490)----|                                              -       D  E  Y  G  G [SEQ ID NO:66]                                         5'   GACGAGTACGGTGGGCTCGAGTGTCG [SEQ ID NO:65]                                3'   CTGCTCATGCCACCCGAGCTCACAGC [SEQ ID NO:81]                                                 Xho1                                                   

Oligonucleotides:

    #37 38mer/0.2um/OFF                                                                       5'CGACACCGCGGCCACCATGAAGCTACTGTCTT                                                                    [SEQ ID NO: 41]                             CTATCG                                                                        #38 28mer/0.2um/OFF 5'CGACAGTCGACCGATACAGTCAACTGTC [SEQ ID NO:42]                                                           #39 34mer/0.2um/OFF                                                          5'CGACACCGCGGCCACCATGGTTTCT                                                   AAGCTGAGC [SEQ ID NO:49]                                                       #40 28mer/0.2um/OFF                                                          5'CGACAGTCGACCAACTTGTGCCGGA                                                   AGG [SEQ ID NO:48]                                                             #43 29mer/0.2um/OFF                                                          5'CGACAGTCGACGCCCCCCCGACCGA                                                   TGTC [SEQ ID NO:61]                                                            #44 26mer/0.2um/OFF                                                          5'CGACACTCGAGCCCACCGTACTCGT                                                   C [SEQ ID NO:62]                 #45 26mer/0.2um/OFF 5'GGCCACCATGC [SEQ ID NO:53]                              #46 18mer/0.2um/OFF 5'TCGAGCATGGTGGCCGC [SEQ ID NO:55]                        #47 27mer/0.2um/OFF 5'TCGACCCTAAGA-(C/A)-GAAGAGAAAGGTAC [SEQ ID NO:56]                                                      #48 27mer/0.2um/OFF                                                          5'TCGAGTACCTTTCTCTTC-(G/T)-                                                   TCTTAGGG [SEQ ID NO:57]    

Example 8. Demonstration of Transcriptional Induction.

Jurkat TAg cells were transfected with the indicated constructs (5 μg ofeach construct) by electroporation (960 μF, 250 v). After 24 hours, thecells were resuspended in fresh media and aliquoted. Half of eachtransfection was incubated with the dimeric FK506 derivative, (Example14) at a final concentration of 1 μM After 12 hours, the cells werewashed and cellular extracts were prepared by repeated freeze-thaw.Chioramphenicol acetyltransferase (CAT) activity was measured bystandard protocols. Molecular Cloning: A Laboratory Manual, Sambrook etal. eds. (1989) CSH Laboratory, pp. 16-59 ff. The data (FIG. 22)demonstrates CAT activity present in 70 μL of extract (total extractvolume was 120 μL) after incubation at 37° C. for 18 hours. The samplesemployed in the assays are as follows:

1. G5E4TCAT (GAL4-CAT reporter plasmid)

2. G5E4TCAT, GAL4-VP16

3. G5E4TCAT, NF3V1E

4. G5E4TCAT, GF2E

5. G5E4TCAT, GF2E, NF3V1E

6. G5E4TCAT, GF3E, NF3V1E

Synthetic Chemistry Examples

As indicated elsewhere, compounds of particular interest at present asoligomerization agents have the following structure:

    linker--{rbm.sub.1,rbm2, . . . rbm.sub.n }.

wherein "linker" is a linker moiety such as described herein which iscovalently linked to "n" (an integer from 2 to about 5, ususally 2 or 3)receptor binding moieties ("rbm"'s) which may be the same or different.As discussed elsewhere herein, the receptor binding moiety is a ligand(or analog thereof) for a known receptor, such as are enumerated inSection V(C), and including FK506, FK520, rapamycin and analogs thereofwhich are capable of binding to an FKBP; as well as cyclosporins,tetracyclines, other antibiotics and macrolides and steroids which arecapable of binding to respective receptors.

The linker is a bi- or multi-functional molecule capable of beingcovalently linked ("--") to two or more receptor binding moieties.Illustrative linker moieties are disclosed in Section VI(A) and in thevarious Examples and include among others C2-C20 alkylene, C4-18azalkylene, C6-C24 N-alkylene azalkylene, C6-C18 arylene, C8-C24ardialkylene and C8-C36 bis-carboxamido alkylene.

These compounds may be prepared using commercially available materialsand/or procedures known in the art. Engineered receptors for thesecompounds may be obtained as described infra. Compounds of particularinterest are those which bind to a receptor with a Kd of less than 10⁻⁶preferably less than about 10⁻⁷ and even more preferably, less than10⁻⁸.

One subclass of oligomerizing agents of interest are those in which oneor more of the receptor binding moieties is FK506, an FK-506-typecompound or a derivative thereof, wherein the receptor binding moietiesare covalently attached to the linker moiety through the allyl group atC21 (using FK506 numbering) as per compound 5 or 13 in FIG. 23A, orthrough the cyclohexyl ring (C29-34), e.g. through the C32 hydroxyl asper compounds 8,16,17 in FIG. 23B. Compounds of this class may beprepared by adaptation of methods disclosed herein, including in theexamples which follow.

Another subclass of oligomerizing agents of interest are those in whichat least one of the receptor binding moieties is FK520 or a derivativethereof, wherein the molecules of FK520 or derivatives thereof arecovalently attached to the linker moiety as in FK1040A or FK 1040B inFIG. 10. Compounds of this class mnay be prepared by adaptation ofScheme 1 in FIG. 10, Scheme 2 in FIGS. 11A and 11B or Scheme 3 in FIG.12 and FIG. 13.

A further subclass of oligomerizing agents of interest are those inwhich at least one of the receptor binding moieties is cyclosporin A ora derivative.

It should be appreciated that these and other oligomerizing agents ofthis invention may be homo-oligomerizing reagents (where the rbm's arethe same) or hetero-oligomerizing agents (where the rbm's aredifferent). Hetero-oligomerizing agents may be prepard by analogy to theprocedures presented herein, including Scheme 3 in FIG. 13 and asdiscussed elsewhere herein.

The following synthetic examples are intended to be illustrative.

A. General Procedures. All reactions were performed in oven-driedglassware under a positive pressure of nitrogen or argon. Air andmoisture sensitive compounds were introduced via syringe or cannulathrough a rubber

B. Physical Data. Proton magnetic resonance spectra (¹ H NMR) wererecorded on Bruker AM-500 (500 MHz), and AM-400 (400 MHz) spectrometers.Chemical shifts are reported in ppm from tetramethylsilane using thesolvent resonance as an internal standard (chloroform, 7.27 ppm). Dataare reported as follows: chemical shift, multiplicity (s=singlet,d=doublet, t=triplet, q=quartet, br=broadened, m=multiplet), couplingconstants (Hz), integration. Low and high-resolution mass spectra wereobtained.

C. Chromatography. Reactions were monitored by thin layer chromatography(TLC) using E. Merck silica gel 60 F glass plates (0.25 mm). Componentswere visualized by illumination with long wave ultraviolet light,exposed to iodine vapor, and/or by dipping in an aqueous ceric ammoniumnmolybdate solution followed by heating. Solvents for chromatography wereHPLC grade. Liquid chromatography was performed using forced flow (flashchromatography) of the indicated solvent system on E. Merck silica gel60 (230-400 mesh).

D. Solvents and Reagents. All reagents and solvents were analyticalgrade and were used as received with the following exceptions.Tetrahydrofuran (THF), benzene, toluene, and diethyl ether weredistilled from sodium metal benzophenone ketyl. Triethylamine andacetonitiie were distilled from calcium hydride. Dichloromethane wasdistilled from phosphorous pentoxide. Dimethylformamide (DMF) wasdistilled from calcium hydride at reduced pressure and stored over 4 Åmolecular sieves.

Preparation of FK506 Derivatives

Example 9. Hydroboration/Oxidation of FK506-TBS₂ (1 to 2).

The hydroboration was performed according to the procedure of Evans(Evans, et al., JACS (1992) 114, 6679; ibid. (1992) 6679-6685). (SeeHarding, et al., Nature (1989) 341, 758 for numbering.) A 10-mL flaskwas charged with 24,32-bis[(tert-butyldimethylsilyl)oxy]-FK506 (33.8 mg,0.033 mmol) and [Rh(nbd)(diphos-4)]BF₄ (3.1 mg, 0.004 mmol, 13 mol %).The orange mixture was dissolved in toluene (2.0 mL) and the solvent wasremoved under reduced pressure over four hours. The flask was carefullypurged with nitrogen and the orangish oil was dissolved in THF (3.0 mL,10 mM final concentration) and cooled to 0° C. with an ice water bath.Catecholborane (98 μL, 0.098 mmol, 1.0M solution in TBF, 3.0 equiv.) wasadded via syringe and the resulting solution was stirred at 0° C. for 45min. The reaction was quenched at 0° C. with 0.2 mL of THF/EtOH (1:1)followed by 0.2 mL of pH 7.0 buffer (Fisher; 0.05M phosphate) then 0.2mL of 30% H₂ O₂. The solution was stirred at room temperature for atleast 12 h. The solvent was removed under reduced pressure and theremaining oil was dissolved in benzene (10 mL) and washed with saturatedaqueous sodium bicarbonate solution (10 mL). The phases were separatedand the aqueous phase was back-extracted with benzene (2×10 mL). Theorganic phases were combined and washed once with saturated aqueoussodium bicarbonate solution (10 mL). The benzene phase was dried withMgSO₄, concentrated, and subjected to flash chromatography (2:1hexane:ethyl acetate) providing the desired primary alcohol as a clear,colorless oil (12.8 mg, 0.012 mmol, 37%).

Preparation of Mixed Carbonate (2 to 3). The preparation of the mixedcarbonate was accomplished by the method of Ghosh (Ghosh, et al.,Tetrahedron Lett. (1992) 33, 2781-2784). A 10-mL flask was charged withthe primary alcohol (29.2 mg, 0.0278 mmol) and benzene (4 mL). Thesolvent was removed under reduced pressure over 60 min. The oil wasdissolved in acetonitrile (2.0 mL, 14 mM final concentration) andstirred at 20° C. as triethylamine (77 μL, 0.56 mmol) was added.N,N'-disuccinimidyl carbonate (36 mg, 0.14 mmol) was added in oneportion and the solution was stirred at 20° C. for 46 h. The reactionmixture was diluted with dichloromethane and washed with saturatedaqueous sodium bicarbonate solution (10 mL). The phases were separatedand the aqueous layer was back-extracted with dichloromethane (2×10 mL).The organic phases were combined and dried (MgSO₄), concentrated, andsubjected to flash chromatography (3:1 to 2:1 to 1:1 hexane:ethylacetate). The desired mixed carbonate was isolated as a clear, colorlessoil (16.8 mg, 0.014 mmol, 51%).

Dimerization of FK506 (3 to 4). A dry, 1-mL conical glass vial (KontesScientific Glassware) was charged with the mixed carbonate (7.3 mg,0.0061 mmol) and acetonitrile (250 μL, 25 mM final concentration).Triethylamine (10 μL, 0.075 mmol) was added followed byp-xylylenediamine (8.3 μL, 0.0027 mmol, 0.32M solution in DMF). Thereaction stirred 22 h at 20° C. and was quenched by dilution withdichloromethane (10 mL). The solution was washed with saturated aqueoussodium bicarbonate solution (10 mL). The phases were separated and theaqueous layer was back-extracted with dichloromethane (2×10 mL). Theorganic phases were combined and dried (MgSO₄), concentrated, andsubjected to flash chromatography (3:1 to 2:1 to 1:1 hexane:ethylacetate) providing the desired protected dimer as a clear, colorless oil(4.3 mg, 1.9 μmol, 70%).

Deprotection of the FK506 Dimer (4 to 5). The protected dimer (3.3 mg,1.4 μmol) was placed in a 1.5-mL polypropylene tube fitted with a spinvane. Acetonitrile (0.5 mL, 3 mM final concentration) was added and thesolution stirred at 20° C. as HF (55 μL, 48% aqueous solution; Fisher)was added. The solution was stirred 18 h at room temperature. Thedeprotected FK506 derivative was then partitioned betweendichloromethane and saturated aqueous sodium bicarbonate in a 15-mL testtube. The tube was vortexed extensively to mix the phases and, afterseparation, the organic phase was removed with a pipet. The aqueousphase was back-extracted with dichloromethane (4×2 mL), and the combinedorganic phases were dried (MgSO₄), concentrated and subjected to flashchromatography (1:1:1 hexane:THF:ether to 1:1 THF:ether) providing thedesired dimer as a clear, colorless oil (1.7 mg, 0.93 μmol, 65%).

Following the above procedure, other monoamines and diamines may beused, such as benzylamine (14) octamethylenediamine,decamethylenediamine, etc.

Example 10. Reduction of FK506 with L-Selectride (FK506 to 6).Danishefsky and coworkers have shown that the treatment of FK506 withL-Selectride provides 22-dihydro-FK506 with a boronate ester engagingthe C24 and C22 hydroxyl groups (Coleman and Danishefsky, Heterocydes(1989) 28, 157-161; Fisher, et al., J. Org. Chem. (1991) 56, 2900-2907).

Preparation of the Mixed Carbonate (6 to 7). A 10-mL flask was chargedwith 22-dihydro-FK506-sec-butylboronate (125.3 mg, 0.144 mmol) andacetonitrile (3.0 mL, 50 mM final concentration) and stirred at roomtemperature as triethylamine (200 μL, 1.44 mmol, 10 equiv.) was added tothe clear solution. N,N'-disuccinimnidyl carbonate (184.0 mg, 0.719mnmol) was added in one portion, and the clear solution was stirred atroom temperature for 44 h. The solution was diluted with ethyl acetate(20 mL) and washed with saturated aqueous sodium bicarbonate (10 mL) andthe phases were separated. The aqueous phase was then back-extractedwith ethyl acetate (2×10 mL), and the organic phases were combined,dried (MgSO₄), and the resulting oil was subjected to flashchromatography (1:1 to 1:2 hexane:ethyl acetate) providing the desiredmixed carbonate as a clear, colorless oil (89.0 mg, 0.088 mmol, 61%).

Dimerization of FK506 Mixed Carbonate (7 to 8). A dry, 1-mL conicalglass vial (Kontes Scientific Glassware) was charged with the mixedcarbonate (15.0 mg, 0.0148 mmol) and dichloromethane (500 μL, 30 mMfinal concentration). The solution was stirred at room temperature astriethylamine (9 μL, 0.067 mmol, 10 equiv.) was added followed byp-xylylenediamnine (0.8 mg, 0.0059 mmol). The reaction stirred 16 h at20° C. and was quenched by dilution with dichloromethane (5 mL). Thesolution was washed with saturated aqueous sodium bicarbonate solution(5 mL). The phases were separated and the aqueous layer wasback-extracted with dichloromethane (2×5 mL). The organic phases werecombined and dried (MgSO₄), concentration, and subjected to flashchromatography (1:1 to 1:2 hexane:ethyl acetate) providing the desireddimer as a clear, colorless oil (7.4 mg, 3.8 pmol, 65%).

Following the above procedure, other, monoamnines, diamines or triaminesmay be used in place of the xylylenediamine, such as benzylamnine (15),octylenediamine, decamethylenediamine (16), bis-p-dibenzylamine,N-methyl diethyleneamnine, tris-aminoethylantine (17),tris-amninopropylamine, 1,3,5-triaminomethylcyclohexane, etc.

Example 11. Oxidative Cleavage and Reduction of FK506 (1 to 9). Theosmylation was performed according to the procedure of Kelly(VanRheenen, et al., Tetrahedron Lett. (1976) 17, 1973-1976). Thecleavage was performed according to the procedure of Danishefsky (Zell,et al., J. Org. Chem. (1986) 51, 5032-5036). The aldehyde reduction wasperformed according to the procedure of Krishnamurthy (J. Org. Chem.,(1981) 46, 4628-4691). A 10 mL flask was charged with24,32-bis[tert-butyldimethylsilyl)oxy]-FK506 (84.4 mg, 0.082 mmol),4-methylmorpholine N-oxide (48 mg, 0.41 mmol, 5 equiv), and THF (2.0 mL,41 mM final concentration). Osmium tetroxide (45 μL, 0.008 mmol, 0.1equiv) was added via syringe. The dear, colorless solution was stirredat room temperature for 5 hr. The reaction was then diluted with 50%aqueous methanol (1.0 mL) and sodium periodate (175 mg, 0.82 mmol, 10equiv) was added in one portion. The cloudy mixture was stirred 40 minat room temperature, diluted with ether (10 mL), and washed withsaturated aqueous sodium bicarbonate solution (5 mL). The phases wereseparated and the aqueous layer was back-extracted with ether (2×5 mL).The combined organic layers were dried (MgSO₄) and treated with solidsodium sulfite (50 mg). The organic phase was then filtered andconcentrated and the oil was subjected to flash chromatography (3:1 to2:1 hexane:ethyl acetate) providing the intermediate, unstable aldehyde(53.6 mg) as a clear, colorless oil. The aldehyde was immediatelydissolved in THF (4.0 mL) and cooled to -78° C. under an atmosphere ofnitrogen, and treated with lithium tris[(3-ethyl-3-pentyl)oxy]aluminumhydride (0.60 mL, 0.082 mmol, 0.14 M solution in THF, 1.0 equiv). Theclear solution was allowed to stir for 10 min at -78° C. then quenchedby dilution with ether (4 mL) and addition of saturated aqueous ammoniumchloride (0.3 mL). The mixture was allowed to warm to room temperatureand solid sodium sulfate was added to dry the solution. The mixture wasthen filtered and concentrated and the resulting oil was subjected toflash chromatography (2:1 hexane:ethyl acetate) giving the desiredalcohol as a dear, colorless oil (39.5 mg, 0.038 mmol, 47%).

Preparation of Mixed Carbonate (9 to 10). The preparation of the mixedcarbonate was accomplished by the method of Ghosh, et al., TetrahedronLett. (1992) 33, 2781-2784). A 10 mL flask was charged with the primaryalcohol (38.2 mg, 0.0369 mmol) and acetonitrile (2.0 mL, 10 mM finalconcentration) and stirred at room temperature as 2,6-lutidine (43 μL,0.37 mmol, 10 equiv) was added. N,N'-disuccinimidyl carbonate (48 mg,0.18 mmol) was added in one portion and the solution was stirred at roomtemperature for 24 h. The reaction mixture was diluted with ether (10mL) and washed with saturated aqueous sodium bicarbonate solution (10mL). The phases were separated and the aqueous layer was back-extractedwith ether (2×10 mL). The organic phases were combined and dried(MgSO₄), concentrated, and subjected to flash chromatography (2:1 to 1:1hexane:ethyl acetate). The desired mixed carbonate was isolated as adear, colorless oil (32.6 mg, 0.028 mmol, 75%).

Preparation of Benzyl Carbamate (10 to 11). A dry, 1 mL conical glassvial (Kontes Scientific Glassware) was charged with the mixed carbonate10 (8.7 mg, 0.0074 mmol) and acetonitrile (500 μL, 15 mM finalconcentration). The solution was stirred at room temperature astriethylamine (10 μL, 0.074 mmol, 10 equiv) was added followed bybenzylamine (1.6 μL, 0.015 mmol, 2 equiv). The reaction stirred 4 h atroom temperature. The solvent was removed with a stream of dry nitrogenand the oil was directly subjected to flash chromatography (3:1 to 2:1hexane:ethyl acetate) providing the desired protected monomer as a dear,colorless oil (6.2 mg, 5.3 μmol, 72%).

The protected monomer (0.2 mg, 5.3 μmol) was placed in a 1.5 mLpolypropylene tube fitted with a spin vane. Acetonitrile (0.5 mL, 11 mMfinal concentration) was added and the solution stirred at roomtemperature as HF (55 μL, 48% aqueous solution; Fisher, 3.0 N finalconcentration) was added. The solution was stirred 18 h at roomtemperature. The deprotected FK506 derivative was then partitionedbetween dichloromethane and saturated aqueous sodium bicarbonate in a 15mL test tube. The tube was vortexed extensively to mix the phases and,after separation, the organic phase was removed with a pipet. Theaqueous phase was back-extracted with dichloromethane (4×2 mL), and thecombined organic phases were dried (MgSO₄), concentrated and subjectedto flash chromatography (1:1 to 0:1 hexane:ethyl acetate) providing forthe desired deprotected benzylcarbamate as a clear, colorless oil (3.9mg, 4.1 μmol, 78%).

By replacing the benzylamine with a diamine such as xylylenediamine(12), hexamethylenediamine, octamethylenediamine, decamethylenediamine(13) or other diamines, dimeric compounds of the subject invention areprepared.

EXAMPLE 12 Preparation of the Mixed Carbonate of FK506 (12)

A 10-mL flask was charged with 24, 32-bis[(tert-butyldimethylsilyl)oxy]-FK506 (339.5 mg., 0.329 mmol),4-methylmorpholine N-oxide (193 mg, 1.64 mmol, 5 equiv), water (0-20 mL)and THF (8.0 mL, 41 mN final concentration). Osmium tetroxide (0.183 mL,0.033 mmol, 0.1 equiv, 0.18 M soln in water) was added via syringe. Theclear, colorless solution was stirred at room temperature for 4.5 h. Thereaction was diluted with 50% aqueous methonol (4.0 mL) and sodiumperiodate (700 mg, 3.29 mmol, 10 equiv) was added in one portion. Thecloudy mixture was stirred 25 min at room temperture, diluted with ether(20 mL), and washed with saturated aqueous sodium bicarbonate solution(10 mL). The phases were separated and the aqueous layer wasback-extracted with ether (2×10 mL). The combined organic layers weredried over MgSO₄ and solid sodium sulfite (50 mg). The organic phase wasthen filtered and concentrated and the resulting aldehyde wasimmediately dissolved in THF (8.0 mL) and cooled to -78° C. under anatmosphere of nitrogen, and treated with lithium tris[(3-ethyl-3-pentyl)oxy] aluminum hydride (2.35 mL, 0.329 mmol, 0.14 Msolution of THF, 1.0 equiv). The dear solution was allowed to stir for60 min at -78° C. (monitored closely by TLC) then quenched at -78° C. bydilution with ether (5 mL) and addition of saturated aqueous ammoniumchloride (0.3 mL). The mixture was allowed to warm to room temperatureand solid sodium sulfate was added to dry the solution. The mixture wasstirred 20 min, filtered, concentrated, and the resulting oil wasimmediately dissolved in acetonitrile (10 mL). To the solution of theresulting primary alcohol in CH₃ CN was added 2,6-lutidine (0.380 mL,3.3. mmol, 10 equiv) and N,N'-disuccinimidyl carbonate (420 mg, 1.65mmol, 5 equiv). The heterogenous mixture was stirred at room temperaturefor 19 h, at which time the solution was diluted with ether (30 mL) andwashed with saturated aqueous sodium bicarbonate (20 mL). The aqueousphase was back-extracted with ether (2×10 mL). The organic phases werecombined and dried (MgSO₄), concentrated, and subjected to flashchromatography (3:1 to 2:1 to 1:1 hexane/ethyl acetate). The desiredmixed carbonate 12 was isolated as a dear, colorless oil (217 mg, 0.184mmol, 56% overall for 4 steps)

EXAMPLE 13 Preparation of 24, 24', 32,32'-Tetrakis[(tert-butyldimethylsilyl)oxy]-FK1012-A

(p-xylylenediamine bridge) A dry, 1-mL conical glass vial was chargedwith the mixed carbonate (23.9 mg, 0.0203 mmol) and acetonitrile (500μL, 41 mM final concentration). Triethylamine (28 μL, 0.20 mmol, 10equiv) was added followed by p-xylylenediamine (46 μL, 0.0101 mmol, 0.22M solution in DMF). The reaction stirred 18 h at room temperature, thesolvent was removed with a stream of dry nitrogen, and the oil wasdirectly subjected to flash chromatography (3:1 to 2:1 to 1:1hexane/ethyl acetate) affording the desired protected dimer as a clear,colorless oil (11.9 mg, 5.3 μmol, 52%)

EXAMPLE 14 Preparation of FK1012-A (p-Xylylenediamine Bridge) (13)

The protected dimer (11.0 mg, 4.9 μmol) was placed in a 1.5-mLpolypropylene tube fitted with a spin vane. Acetonitrile (0.50 mL, 10 mMfinal concentration) was added, and the solution stirred at 20° C. as HF(55 μL, 48% aqueous solution; Fisher, 3.0 N final. concentration) wasadded. The solution was stirred 16 h at room temperature. Thedeprotected FK506 derivative was then partitioned betweendichloromethane and saturated aqueous sodium bicarbonate in a 15-mL testtube. The tube was vortexed extensively to mix the phases and, afterseparation, the organic phase was removed with a pipet. The aqueousphase was back-extracted with dichloromethane (4×2 mL), and the combinedorganic phases were dried (MgSO₄), concentrated and subjected to flashchromatography (1:1:1 hexane/THF/ether to 1:1 THF/ether) providingFK1012-A as a dear, colorless oil (5.5 mg, 3.0 μmol, 63%).

EXAMPLE 15 Preparation of 24, 24', 32,32'-Tetrakis[(ter-butyldimethylsilyl)oxy]-FK1012-B (DiaminodecaneBridge)

A dry, 1-mL conical glass vial was charged with the mixed carbonate(53.3 mg, 0.0453 mmol) and acetonitrile (2.0 mL, 11 m M finalconcentration). Triethylamine (16 μL, 0.11 mmol, 5 equiv) was addedfollowed by diaminodecane (61 μL, 0.0226 mmol, 0.37 M solution in DMF).The reaction stirred 12 h at room temperature, the solvent was removedwith a stream of dry nitrogen, and the oil was directly subjected toflash chromatography (3:1 to 2:1 to 1:1 hexane/ethyl acetate) affordingthe desired protected dimer as a clear, colorless oil (18.0 mg, 7.8μmol, 35%).

EXAMPLE 16 Preparation of FK1012-B (Diaminodecane-1,10 Bridge) (14)

The protected dimer (18.0 mg, 7.8 ,μmol) was placed in a 1.5-mLpolypropylene tube fitted with a stirring flea. Acetonitrile (0.45 mL,16 mM final concentration) was added, and the solution sitrred at roomtemperature as HF (55 μL, 48% aqueous solution; Fisher, 3.6 N finalconcentration) was added. The solution was stirred 17 h at 23° C. Theproduct FK1012-B was then partitioned between dichloromethane andsaturated aqueous sodium bicarbonate in a 15-mL test tube. The tube wasvortexed extensively to mix the phases and, after separation, theorganic phase was removed with a pipet The aqueous phase wasback-extracted with dichloromethane (4×2 mL), and the combined organicphases were dried (MgSO₄), concentrated and subjected to flashchromatography (100% ethyl acetate to 20:1 ethyl acetate/methanol)affording FK1012-B as a dear, colorless oil (5.3 mg, 2.9 μmol, 37%).

EXAMPLE 17 Preparation of 24, 24', 32,32'-Tetrakis[(tert-butyldimethylsilyl)oxy]-FK1012-C(his-p-Aminomethylbenzoyl Diaminodecane Bridge)

A dry 25-mL tear-shaped flask was charged with the diamine linker (15.1mg, 0.0344 mmol) and 1.0 mL of DMF. In a separate flask, the mixedcarbonate and triethylamine (0.100 mL, 0.700 mmol, 20 equiv) weredissolved in 2.0 mL of dichloromethane then added slowly (4×0.50 mL) tothe stirring solution of his-p-aminomethylbenzoyl, diaminodecane-1,10.The flask containing the mixed carbonate 12 was washed withdichloromethane (2×0.50 mL) to ensure complete transfer of the mixedcarbonate 12. The reaction stirred 16 h at 23° C., the solvent wasremoved with a stream of dry nitrogen, and the oil was directlysubjected to flash chromatography (1:1 to 1:2 hexane/ethyl acetate) toafford the desired protected dimer as a clear, colorless oil (29.6 mg,11.5 μmol, 34%).

EXAMPLE 18 Preparation of FK1012-C (15)

The protected dimer (29.6 mg, 11.5 μmol) (17) was placed in a 1.5-mLpolypropylene tube fitted with a stirring flea. Acetonitrile (0.45 mL,23 mM final concentration) was added, and the solution stirred at roomtemperature as HF (55 μL, 48% aqueous solution; Fisher, 3.6 N finalconcentration) was added. The solution was stirred 17 h at roomtemperature. The desired symmetrical dimer was then partitioned betweendichloromethane and saturated aqueous sodium bicarbonate in a 15-mL testtube. The tube was vortexed extensively to mix the phases and, afterseparation, the organic phase was removed with a pipet. The aqueousphase was back-extracted with dichloromethane (4×2 mL), and the combinedorganic phases were dried (MgSO₄), concentrated and subjected to flashchromatography (100% ethyl acetate to 15:1 ethyl acetate/methanol)affording FK1012C as a clear, colorless oil (11.5 mg, 5.5 μmol, 47%).

Preparation of CsA Derivatives EXAMPLE 19 MeBmt(OAc)--OH¹ CsA (2)

MeBmt(OAc)--OAc--CsA (1) (161 mg, 124 mmol) (see Eberle and Nuninger, J.Org. Chem. (1992) 57, 2689) was dissolved in Methanol (10 mL). KOH (196mg) was dissolved in water (8 mL). 297 mL of the KOH solution (0.130mmol, 1.05 eq.) was added to the solution of (1) in MeOH. This newsolution was stirred at room temperature under an inert atmosphere for 4hours at which time the reaction was quenched with acetic acid (2 mL).The reaction mixture was purified by reversed phase HPLC using a 5 cm×25cm, 12 μL, 100 A, C18 column at 70° C. eluting with 70% acetonitrile/H₂O containing 0.1% (v/v) Trifluoroacetic acid to give 112 mg (72%) of thedesired monoacetate (2).

MeBmt(OAc)--OCOIm¹ CsA (3)

MeBmt(OAc)--OH¹ CsA (2) (57 mg, 45.5 μmol) and carbonyldiimidazole (15mg, 2 eq., 91 μmol.) were transferred into a 50 mL round bottom flaskand dissolved in dry THF (6 mL). Diisopropylethylamine (32 μL, 4 eq.,182 μmol) was added and then the solvent was removed on a rotaryevaporator at room temperature. The residue was purified by flashchromatography on silica gel using ethyl acetate as eluent to give 45 mg(73%) of the desired carbamate (3).

Tris-2-aminoethyl)amine CsA Trimer Triacetate (6)

MeBmt(OAc)--OCOIm¹ -CsA (3) (7.5 mg, 5.54 μmol, 3.1 eq.) was dissolvedin THF (100 μL). Diisopropylethylamine (62 μL, 5 eq., 8.93 μmol of asolution containing 100 μL of amine in 4 mL THF) was added followed bytris(2-aminoethyl)amine (26 μL, 1.79 μmol, 1 eq. of a solutioncontaining 101 mg of tris-amine in 10 mL THF). This solution was allowedto stir under N₂ atmosphere for 5 days. The reaction mix was evaporatedand then purified by flash chromatography on silica gel using 0-5%methanol in chloroform to give 41 mg of desired product (6).

EXAMPLE 20 Diaminodecane CsA Dimer (8)

Solid Na metal (200 mg, excess) was reacted with dry methanol (10 mL) at0° C. Diaminodecane CsA Dimer Diacetate (5) (4.0 mg) was dissolved inMeOH (5 mL). 25 mL of the NaOMe solution was added to the solution of(5). After 2.5 hours of stirring at room temperature under an inertatmosphere, the solution was quenched with acetic acid (2 mL) and theproduct was purified by reversed phase HPLC using a 5 mm×25 mm, 12 I,100 A, C18 column at 70° C. eluting with 70-95% acetonitrile/H₂ O over20 minutes containing 0.1% (v/v) Trifluoroacetic acid to give 2.5 mg(60%) of the desired diol.

The diaminodecane CsA Dimer Diacetate (5) was prepared by replacing thetris(2-aminoethyl)amine with 0.45 eq. of 1,10-aminodecane.

EXAMPLE 21 p-Xylylenediamine CsA Dimer (4)

The p-xylene diamine CsA Dimer (4) was prepared by replacing thetris(2-aminoethyl)amino with 0.45 eq. of p-xylylene diamine.

Following procedures described in the literature other derivatives ofcyclophilin are prepared by linking at a site other than the 1(MeBmt 1)site.

Position 8 D-isomer analogues are produced by feeding the producingorganism with the D-amino analogue to obtain incorporation specificallyat that site. See Patchett, et al, J. Antibiotics (1992) 45, 943(β-MeSO)D-Ala⁸ -CsA); Traber, et al., ibid. (1989) 42, 591). Theposition 3 analogues are prepared by poly-lithiation/alkylation of CsA,specifically at the -carbon of Sac3. See Wenger, Transplant Proceeding(1986) 18, 213, supp. 5 (for cyclophilin binding and activity profiles,particularly D-MePhe³ -CsA); Seebach, U.S. Pat. No. 4,703,033, issuedOct. 27, 1987 (for preparation of derivatives).

Instead of cyclosporin A, following the above-described procedures,other naturally-occurring variants of CsA may be multimerized for use inthe subject invention.

EXAMPLE 22 (A) Structure-Based Design and Synthesis of FK1012-"Bump"Compounds and FKBP12s with Compensatory Mutations

Substituents at C9 and C10 of FK506, which can be and have been accessedby synthesis, clash with a distinct set of FKBP12 sidechain residues.Thus, one class of mutant receptors for such ligands should containdistinct modifications, one creating a compensatory hole for the C10substituent and one for the C9 substituent. Carbon 10 was selectivelymodified to have either an N-acetyl or N-formyl group projecting fromthe carbon (vs. a hydroxyl group in FK506). The binding properties ofthese derivatives clearly reveal that these C10 bumps effectivelyabrogate binding to the native FKBP12. FIG. 23 depicts schemes for thesynthesis of FK506-type moieties containing additional C9 bumps. Byassembling such ligands with linker moieties of this invention one canconstruct HED and HOD (and antagonist) reagents for chimeric proteinscontaining corresponding binding domains bearing compensatory mutations.An illustrative HED reagent is depicted in FIG. 23 that containsmodifications at C9 and C10.

This invention thus encompasses a class of FK-506-type compoundscomprising an FK-506-type moiety which contains, at one or both of C9and C10, a functional group comprising --OR, --R, --(CO)OR, --NH(CO)H or--NH(CO)R, where R is substituted or unsubstituted, alkyl or arylalkylwhich may be straightchain, branched or cyclic, including substituted orunsubstituted peroxides, and carbonates. "FK506-type moieties" includeFK506, FK520 and synthetic or naturally occurring variants, analogs andderivatives thereof (including rapamycin) which retain at least the(substituted or unsubstituted) C2 through C15 portion of the ringstructure of FK-506 and are capable of binding with a natural ormodified FKBP, preferably with a Kd value below about 10⁻⁶.

This invention further encompasses homo- and hetero-dimers and higherorder oligomers containing one or more of such FK-506-type compoundscovalently linked to a linker moiety of this invention. Monomers ofthese FK-506-type compounds are also of interest, whether or notcovalently attached to a linker moiety or otherwise modified withoutabolishing their binding affinity for the corresponding FKBP. Suchmonomeric compounds may be used as oligomerization antagonist reagents,i.e., as antagonists for oligomerizing reagents based on a likeFK-506-type compound. Preferably the compounds and oligomers comprisingthem in accordance with this invention bind to natural, or preferablymutant, FKBPs with an affinity at least 0.1% and preferably at leastabout 1% and even more preferably at least about 10% as great as theaffinity of FK506 for FKBP12. See e.g. Holt et al, infra.

Receptor domains for these and other ligands of this invention may beobtained by structure-based, site-directed or random mutagenesismethods. We contemplate a family of FKBP12 moieties which contain Val,Ala, Gly, Met or other small amino acids in place of one or more ofTyr26, Phe36, Asp37, Tyr82 and Phe99 as receptor domains for FK506-typeand FK-520-type ligands containing modifications at C9 and/or C10.

Site-directed mutagenesis maybe conducted using the megaprimermutagenesis protocol (see e.g., Sakar and Sommer, BioTechniques 8 4(1990): 404-407). cDNA sequencing is performed with the Sequenase kitExpression of mutant FKBP12s may be carried out in the plasmid pHN1⁺ inthe E. coli strain XA90 since many FKBP12 mutants have been expressed inthis system efficiently. Mutant proteins may be conveniently purified byfractionation over DE52 anion exchange resin followed by size exclusionon Sepharose as described elsewhere. See e.g. Aldape et al, J Biol Chem267 23 (1992): 16029-32 and Park et al, J Biol Chem 267 5 (1992):3316-3324. Binding constants may be readily determined by one of twomethods. If the mutant FKBPs maintain sufficient rotamase activity, thestandard rotamase assay may be utilized. See e.g., Galat et al,Biochemistry 31 (1992): 2427-2434. Otherwise, the mutant FKBP12s may besubjected to a binding assay using LH20 resin and radiolabeledT2-dihydroFK506 and T2-dihyroCsA that we have used previously with FKBPsand cyclophilins. Bierer et al, Proc. Natl. Acad. Sci. U.S.A. 87 4(1993): 555-69.

(B) Selection of Compensatory Mutations in FKBP12 for Bump-FK506s Usingthe Yeast Two-Hybrid System

One approach to obtaining variants of receptor proteins or domains,including of FKBP12, is the powerful yeast "two-hybrid" or "interactiontrap" system. The two-hybrid system has been used to detect proteinsthat interact with each other. A "bait" fusion protein consisting of atarget protein fused to a transcriptional activation domain isco-expressed with a cDNA library of potential "hooks" fused to aDNA-binding domain. A protein-protein (bait-hook) interaction isdetected by the appearance of a reporter gene product whose synthesisrequires the joining of the DNA-binding and activation domains. Theyeast two-hybrid system mentioned here was originally developed byElledge and co-workers. Durfee et al, Genes & Development 7 4 (1993):555-69 and Harper et al, Cell 75 4 (1993): 805-816.

Since the two-hybrid system per se cannot provide insights intoreceptor-ligand interactions involving small molecule, organic ligands,we have developed a new, FK1012-inducible transcriptional activationsystem (discussed below). Using that system one may extend the twohybrid system so that small molecules (e.g., FK506s or FK1012s orFK506-type molecules of this invention) can be investigated. One firstgenerates a cDNA library of mutant FKBPs (the hooks) with mutations thatare regionally localized to sites that surround C9 and C10 of FK506. Forthe bait, two different strategies may be pursued. The first uses theability of FK506 to bind to FKBP12 and create a composite surface thatbinds to calcineurin. The sequence-specific transcriptional activator isthus comprised of: DNA-binding domain-mutantFKBP12--bump-FK506--calcineurin A-activation domain (where -- refers toa noncovalent binding interaction). The second strategy uses the abilityof FK1012s to bind two FKBPs simultaneously. A HED version of an FK1012may be used to screen for the following ensemble: DNA-bindingdomain-mutant FKBP12--bump-FK506-normal FK506--wildtypeFKBP12-activation domain.

1. Calcineurin-Gal4 activation domain fusion as a bait

A derivative of pSE1107 that contains the Gal4 activation domain andcalcineurin A subunit fusion construct has been constructed. Its abilityto act as a bait in the proposed manner has been verified by studiesusing the two-hybrid system to map out calcineurin's FKBP-FK506 bindingsite.

2. hFKBP12-Gal4 activation domain fusion as a bait

hFKBP12 cDNA may be excised as an EcoRI-HindIII fragment that covers theentire open reading frame, blunt-ended and ligated to the blunt-ended)ho I site of pSE1107 to generate the full-length hFKBP-Gal4 activationdomain protein fusion.

3. Mutant hFKBP12 cDNA libraries

hFKBP12 may be digested with EcoRI and HindIII, blunted and cloned intopAS1 (Durfee et al, supra) that has been cut with NcoI and blunted. Thisplasmid is further digested with NdeI to eliminate the NdeI fragmentbetween the NdeI site in the polylinker sequence of pAS1 and the 5' endof hFKBP12 and religated. This generated the hFKBP12-Gal4 DNA bindingdomain protein fusion. hFKBP was reamplified with primers #11206 [SEQ IDNO:67] and #11210 [SEQ ID NO:75], Primer Table:

    11206                NdeI                                                       SNdFK: 5'-GGAATTC CAT ATG GGC GTG CAG G-3'                                                 H   M   G   V   Q                                                11207          SmaI                                                           3SmFK37: 5'-CTGTC CCG GGA NNN NNN NNN TTT CTT TCC ATC TTC AAG C-3'                                                                  R   S   X   X   X                                                   K   K   G   D   E   L                                                        11208          SmaI                3SmFK27: 5'-CTGTC CCG GGA GGA ATC AAA TTT CTT TCC ATC TTC AAG CAT                                                                   R   S   S   D   F                                                   K   K   G   D   E   L   M                                                        NNN NNN NNN GTG CAC CAC                                                  GCA GG-3'                                X   X   X   H   V   V   C                                                11209         BamHI                                                           3BmFK98: 5'-CGC GGA TCC TCA TTC CAG TTT TAG AAG CTC CAC ATC NNN                                                                          END  E   L                                                   K   L   L   E   V   D   X                                                          NNN NNN AGT GGC ATG                                                      TGG-3'                                   X   X   T   A   H   P                                                    11210         BamHI                                                           3BmFK: 5'-CGC GGA TCC TCA TTC CAG TTT TAG AAG C-3'                                            END  E   L   K   L   L                                      Primer Table [SEQ ID NOS: 67-76]:     Primers used in the construction of      regionally localized hFKBP12 cDNA library for use in screening                for compensatory mutations.                                              

Mutant hFKBP12 cDNA fragments were then prepared using the primerslisted below that contain randomized mutant sequences of hFKBP atdefined positions by the polymerase chain reaction, and were insertedinto the Gal4 DNA binding domain-hFKBP(NdeI/BamHI) construct.

4. Yeast strain

S. cerevisiae Y153 carries two selectable marker genes(his3/β-galctosidase) that are integrated into the genome and are drivenby Gal4 promoters. (Durfee, supra.)

Using Calcineurin-Gal4 Activation Domain as Bait

The FKBP12-FK506 complex binds with high affinity to calcineurin, a type2B protein phosphatase. Since we use C9- or C10 -bumped ligands to serveas a bridge in the two-hybrid system, only those FKBPs from the cDNAlibrary that contain a compensatory mutation generate a transcriptionalactivator. For convenience, one may prepare at least three distinctlibraries (using primers 11207-11209, Primer Table) that will eachcontain 8,000 mutant FKBP12s. Randomized sites were chosen by inspectingthe FKBP12-FK506 structure, which suggested clusters of residues whosemutation might allow binding of the offending C9 or C10 substituents onbumped FK506s. The libraries are then individually screened using bothC9- and C10-bumped FK506s. The interaction between a bumped-FK506 and acompensatory hFKBP12 mutant can be detected by the ability of host yeastto grow on his drop-out medium and by the expression of β-galactosidasegene. Since this selection is dependent on the presence of thebumped-FK506, false positives can be eliminated by substractivescreening with replica plates that are supplemented with or without thebumped-FK506 ligands.

Using hFKBP12-Gal4 Activation Domain as Bait

Using the calcineurin A-Gal4 activation domain to screen hFKBP12 mutantcDNA libraries is a simple way to identify compensatory mutations onFKBP12. However, mutations that allow bumped-FK506s to bind hFKBP12 maydisrupt the interaction between the mutant FKBP12--bumped-FK506 complexand calcineurin. If the initial screening with calcineurin as a baitfails, the wildtype hFKBP12-al4 activation domain will instead be used.An FK1O12 HED reagent consisting of: native-FK506-bumped-FK506 (FIG. 16)may be synthesized and used as a hook. The FK506 moiety of the FK1012can bind the FKBP12Gal4 activation domain. An interaction between thebumped-FK506 moiety of the FK1012 and a compensatory mutant of FKBP12will allow host yeast to grow on his drop-out medium and to expressβ-galactosidase. In this way, the selection is based solely on theability of hFKBP12 mutant to interact with the bumped-FK506. The samesubstractive screening strategy can be used to eliminate falsepositives.

In addition to the in vitro binding assays discussed earlier, an in vivoassay may be used to determine the binding affinity of the bumped-FK506sto the compensatory hFKBP12 mutants. In the yeast two-hybrid system,β-gal activity is determined by the degree of interaction between the"bait" and the "prey". Thus, the affinity between the bumped-FK506 andthe compensatory FKBP12 mutants can be estimated by the correspondingβ-galactosidase activities produced by host yeasts at different HED(native-FK506-bumped-FK506) concentrations.

Using the same strategy, additional randomized mutant FKBP12 cDNAlibraries may be created in other bump-contact residues withlow-affinity compensatory FKBP12 mutants as templates and may bescreened similarly.

Phage Display Screening for High-Affinity Compensatory FKBP Mutations

Some high-affinity hFKBP12 mutants for bump-FK506 may contain severalcombined point mutations at discrete regions of the protein. The size ofthe library that contains appropriate combined mutations can be toolarge for the yeast two-hybrid system's capacity (e.g., >10⁸ mutations).The use of bacteriophage as a vehicle for exposing whole functionalproteins should greatly enhance the capability for screening a largenumbers of mutations. See e.g. Bass et al, Proteins: Structure, Function& Genetics 8 4 (1990): 309-14; McCafferty et al, Nature 348 6301 (1990):552-4; and Hoogenboom, Nucl Acids Res 19 15 (1991): 4133-7. If thedesired high-affinity compensatory mutants is not be identified with theyeast two-hybrid system, a large number of combined mutations can becreated on hFKBP12 with a phage vector as a carrier. The mutant hFKBP12fusion phages can be screened with bumped-FK506-Sepharose as an affinitymatrix, which can be synthesized in analogy to our original FK506-basedaffinity matrices. Fretz et al, J Am Chem Soc 113 4 (1991): 1409-1411.Repeated rounds of binding and phage amplification should lead to theidentification of high-affinity compensatory mutants.

(C) Synthesis of "Bumped (CsA)2s": Modification of MeVal(11)CsA

As detailed above, we have demonstrated the feasibility of usingcyclophilin as a dimerization domain and (CsA)2 as a HOD reagent in thecontext of the cell death signaling pathway. However, to furtheroptimize the cellular activity of the (CsA)2 reagent one may rely uponsimilar strategies as described with FK1012s. Thus, modified (bumped)CsA-based oligomerizing reagents should be preferred in applicationswhere it is particularly desirable for the reagent to be able todifferentiate its target, the artificial protein constructs, fromendogenous cyclophilins.

One class of modified CsA derivatives of this invention are CsA analogsin which (a) NMeVal11 is replaced with NMePhe (which may be substitutedor unsubstituted) or NMeThr (which may be unsubstituted or substitutedon the threonine betahydroxyl group) or (b) the pro-S methyl group ofNMeVal11 is replaced with a bulky group of at least 2 carbon atoms,preferably three or more, which may be straight, branched and/or containa cyclic moiety, and may be alkyl (ethyl, or preferably propyl, butyl,including t-butyl, and so forth), aryl, or arylalkyl. These compoundsinclude those CsA analogs which contain NMeLeu, NMeIle, NMePhe orspecifically the unnatural NMe[betaMePhe], in place of MeVal11. The"(b)" CsA compounds are of formula 2 where R represents a functionalgroup as discussed above. ##STR1##

This invention further encompasses homo- and hetero dimers and higherorder oligomers containg one or more such CsA analogs. Preferably thecompounds and oligomers comprising them in accordance with thisinvention bind to natural, or preferably mutant, cyclophilin proteinswith an affinity at least 0.1% and preferably at least about 1% and evenmore preferably at least about 10% as great as the affinity of CsA forcyclophilin.

A two step strategy may be used to prepare the modified [MeVal¹¹ ]CsAderivatives starting from CsA. In the first step the residue MeVal11 isremoved from the macrocycle. In the second step a selected amino acid isintroduced at the (former) MeVal11 site and the linear peptide iscyclized. The advantage of this strategy is the ready access to severalmodified [MeVal¹¹ ]CsA derivatives in comparison with a total synthesis.The synthetic scheme is as follows: ##STR2##

To differentiate the amide bonds, an N,O shift has been achieved betweenthe amino and the hydroxyl groups from MeBmt1 to give IsoCsA (Ruegger etal, Helv Chim Acta 59 4 (1976): 1075-92) (see scheme above). Thereaction was carried out in THF in the presence of methanesulfonic acid.(Oliyai et al, Pharm Res 9 5 (1992): 617-22). The free amine wasprotected with an acetyl group with pyridine and acetic anhydride in aone-pot procedure. The overall yield of the N-acetyl protected IsoCsA is90%. The ester MeBmt1-MeVal11 bond is then reduced selectively in thepresence of the N-methyl amide bonds, e.g. using DIBAL-H. The resultingdiol is then transformed to the corresponding di-ester with anotheracid-induced N,O shift. This will prepare both the N-acetyl group andMeVal11 residues for removal through hydrolysis of the newly formedesters with aqueous base.

After protection of the free amino group the new amino acid residue isintroduced e.g. with the PyBrop coupling agent Deprotection andcyclization of the linear peptide with BOP in presence of DMAP (Albergand Schreiber, Science 262 5131 (1993): 248-250) completes the synthesisof 2. The binding of bumped-CsAs to cyclophilins can be evaluated by thesame methods described for FK506s and FK1012s. Once cyclophilins areidentified with compensatory mutations, bumped (CsA)2 HED and HODreagents may be synthesized according to the methods discussedpreviously. Of particular interest are bumped CsA compounds which canform dimers which themselves can bind to a cyclophilin protein with 1:2stoichiometry. Homo dimers and higher order homo-oligomers, heterodimersand hetero-higher order oligomers containing at least one such CsA ormodified CsA moiety may be designed and evaluated by the methodsdeveloped for FK1012A and (CsA)2, and optimize the linker element inanalogy to the FK1012 studies.

Mutant cyclophilins that bind our position 11 CsA variants (2) byaccomodating the extra bulk on the ligand may be now be prepared.Cyclophilins with these compensatory mutations may be identified throughthe structure-based site-directed and random mutagenesis/screeningprotocols described in the FK1012 studies.

It is evident from the above results, that the subject method andcompositions provide for great versatility in the production of cellsfor a wide variety of purposes. By employing the subject constructs, onecan use cells for therapeutic purposes, where the cells may remaininactive until needed, and then be activated by administration of a safedrug. Because cells can have a wide variety of lifetimes in a host,there is the opportunity to treat both chronic and acute indications soas to provide short- or long-term protection In addition, one canprovide for cells which will be directed to a particular site, such asan anatomic site or a functional site, where therapeutic effect may beprovided.

Cells can be provided which will result in secretion of a wide varietyof proteins, which may serve to correct a deficit or inhibit anundesired result, such as activation of cytolytic cells, to inactivate adestructive agent, to kill a restricted cell population, or the like. Byhaving the cells present in the host over a defined period of time, thecells may be readily activated by taking the drug at a dose which canresult in a rapid response of the cells in the host. Cells can beprovided where the expressed chimeric receptor is intracellular,avoiding any immune response due to a foreign protein on the cellsurface. Furthermore, the intracellular chimeric receptor proteinprovides for efficient signal transduction upon ligand binding,apparently more efficiently than the receptor binding at anextracellular receptor domain.

By using relatively simple molecules which bind to chimeric membranebound receptors, resulting in the expression of products of interest orinhibiting the expression of products, one can provide for cellulartherapeutic treatment. The compounds which may be administered are safe,can be administered in a variety of ways, and can ensure a very specificresponse, so as not to upset homeostasis.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                  - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 81                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - Met Gly Ser Ser Lys Ser Lys Pro Lys Asp Pr - #o Ser Gln Arg               1               5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - GTTAAGTTAA C               - #                  - #                      - #       11                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - TGACTCAGCG C               - #                  - #                      - #       11                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 6..11                                                           (D) OTHER INFORMATION: - #/note= "Sac II restriction site."          - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #signal                                           (B) LOCATION: 12..16                                                          (D) OTHER INFORMATION: - #/note= "Kozak sequence."                   - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 17..31                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 17..33                                                          (D) OTHER INFORMATION: - #/note= "Region of homology with                          target se - #quence."                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - CGACACCGCG GCCACC ATG GCC ACA ATT GGA GC   - #                  - #             33                                                                                       - #Met Ala Thr Ile Gly                                                        - # 1               5                                        - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - Met Ala Thr Ile Gly                                                       1               5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 6..11                                                           (D) OTHER INFORMATION: - #/note= "Xho I restriction site."           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 12..27                                                          (D) OTHER INFORMATION: - #/note= "Region of homology with                          target se - #quence."                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - CGACACTCGA GAGCCCATGA CTTCTGG          - #                  - #                 27                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..4                                                            (D) OTHER INFORMATION: - #/note= "Translation product of                           complement - #of SEQ ID NO:6, bases 9 to 20."                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - Ser Trp Ala Leu                                                           1                                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 6..11                                                           (D) OTHER INFORMATION: - #/note= "Xho I restriction site."           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 12..41                                                          (D) OTHER INFORMATION: - #/note= "Region of homology with                          target se - #quence."                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 9..41                                                  - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 28                                                              (D) OTHER INFORMATION: - #/note= "A to G."                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - CGACACTC GAG CTC TGC TAC TTG CTA GGT GGA ATC - #CTC TTC                    - #  41                                                                              Glu Leu Cys Tyr Leu L - #eu Gly Gly Ile Leu Phe                                1        - #       5           - #       10                          - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - Glu Leu Cys Tyr Leu Leu Gly Gly Ile Leu Ph - #e                           1               5  - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 3..8                                                            (D) OTHER INFORMATION: - #/note= "Eco RI restriction site."          - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 9..24                                                           (D) OTHER INFORMATION: - #/note= "Region of homology with                          target se - #quence."                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 24                                                              (D) OTHER INFORMATION: - #/note= "G to C."                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #signal                                           (B) LOCATION: complement - #(9..11)                                           (D) OTHER INFORMATION: - #/note= "Translational stop encoded                       in comple - #mentary strand."                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - GCGAATTCTT AGCGAGGGGC CAGC          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..4                                                            (D) OTHER INFORMATION: - #/note= "Translational product of                         complement - #to SEQ ID NO:10, bases 12 to 23."                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - Leu Ala Pro Arg                                                           1                                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 3..8                                                            (D) OTHER INFORMATION: - #/note= "Eco RI restriction."               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 12..17                                                          (D) OTHER INFORMATION: - #/note= "Sal I restriction site."           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #signal                                           (B) LOCATION: complement - #(9..11)                                           (D) OTHER INFORMATION: - #/note= "Translational stop signal                        encoded o - #n complementary strand."                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 18..33                                                          (D) OTHER INFORMATION: - #/note= "Region of homology with                          target se - #quence."                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - GCGAATTCTT AGTCGACGCG AGGGGCCAGG GTC       - #                  - #             33                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..4                                                            (D) OTHER INFORMATION: - #/note= "Translational product of                         complement - #to SEQ ID NO:12, bases 18 to 29."                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - Leu Ala Pro Arg                                                           1                                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 4..9                                                            (D) OTHER INFORMATION: - #/note= "Xho I restriction site."           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #/note= "T to G."                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 4..25                                                           (D) OTHER INFORMATION: - #/note= "Region of homology with                          target se - #quence."                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 10..24                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - GGGCTCGAG CTC GGC TAC TTG CTA G      - #                  - #                   25                                                                               Leu Gly Tyr Leu Leu                                                            1       - #        5                                                - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:15:                    - - Leu Gly Tyr Leu Leu                                                       1               5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 6..11                                                           (D) OTHER INFORMATION: - #/note= "Xho I restriction site."           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 12..26                                                          (D) OTHER INFORMATION: - #/note= "Region of homology with                          target se - #quence."                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - CGACACTCGA GGTGACGGAC AAGGTC          - #                  - #                  26                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 6..11                                                           (D) OTHER INFORMATION: - #/note= "Sal I restriction site."           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 12..26                                                          (D) OTHER INFORMATION: - #/note= "Region of homology with                          target se - #quence."                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - CGACAGTCGA CCCAATCAGG GACCTC          - #                  - #                  26                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: - #/note= "Xho I restriction site."           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 10..15                                                          (D) OTHER INFORMATION: - #/note= "Bsi WI restriction site."          - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 6..32                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - - TCGAG TAT CCG TAC GAC GTA CCA GAC TAC GCA - #G                  - #             33                                                                           Tyr Pro Tyr Asp Val Pro Asp - #Tyr Ala                                         1           - #    5                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - - Tyr Pro Tyr Asp Val Pro Asp Tyr Ala                                        1               5                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: - #/note= "Sal I restriction site."           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - - TCGACTGCGT AGTCTGGTAC GTCGTACGGA TAC       - #                  - #             33                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: - #/note= "Sal I restriction site."           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                              - - TCGACTATCC GTACGACGTA CCAGACTACG CAC       - #                  - #             33                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: - #/note= "Xho I restriction site."           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                              - - TCGAGTGCGT AGTCTGGTAC GTCGTACGGA TAG       - #                  - #             33                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 80 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 6..11                                                           (D) OTHER INFORMATION: - #/note= "Sac II restriction site."          - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #signal                                           (B) LOCATION: 12..16                                                          (D) OTHER INFORMATION: - #/note= "Kozak sequence."                   - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #signal                                           (B) LOCATION: 17..58                                                          (D) OTHER INFORMATION: - #/note= "Myristoylation signal."            - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 59..64                                                          (D) OTHER INFORMATION: - #/note= "Xho I restriction site."           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 65..80                                                          (D) OTHER INFORMATION: - #/note= "Zeta homology."                    - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 17..79                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                              - - CGACACCGCG GCCACC ATG GGG AGT AGC AAG AGC AAG - #CCT AAG GAC CCC             49                                                                                          - #Met Gly Ser Ser Lys Ser Lys Pro Lys Asp P - #ro                            - # 1               5  - #                10                 - - AGC CAG CGC CTC GAG AGG AGT GCA GAG ACT G - #                  - #              80                                                                     Ser Gln Arg Leu Glu Arg Ser Ala Glu Thr                                                    15     - #             20                                         - -  - - (2) INFORMATION FOR SEQ ID NO:24:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                              - - Met Gly Ser Ser Lys Ser Lys Pro Lys Asp Pr - #o Ser Gln Arg Leu Glu       1               5  - #                10  - #                15               - - Arg Ser Ala Glu Thr                                                                  20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:25:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 12..26                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 6..11                                                           (D) OTHER INFORMATION: - #/note= "Xho I restriction site."           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 12..27                                                          (D) OTHER INFORMATION: - #/note= "Region of homology with                          target se - #quence."                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                              - - CGACACTCGA G GAG CTC TGT GAC GAT G     - #                  - #                 27                                                                                  Glu Leu Cys - #Asp Asp                                                         1    - #           5                                             - -  - - (2) INFORMATION FOR SEQ ID NO:26:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                              - - Glu Leu Cys Asp Asp                                                       1               5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:27:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 6..11                                                           (D) OTHER INFORMATION: - #/note= "Xho I restriction site."           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 12..41                                                          (D) OTHER INFORMATION: - #/note= "Region of homology with                          target se - #quence."                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 27..29                                                          (D) OTHER INFORMATION: - #/note= "GAT to AAG."                       - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 9..41                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                              - - CGACACTC GAG CTC TGC TAC TTG CTA AAG GGA ATC - #CTC TTC                    - #  41                                                                              Glu Leu Cys Tyr Leu L - #eu Lys Gly Ile Leu Phe                                1        - #       5           - #       10                          - -  - - (2) INFORMATION FOR SEQ ID NO:28:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                              - - Glu Leu Cys Tyr Leu Leu Lys Gly Ile Leu Ph - #e                           1               5  - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:29:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 44 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 6..11                                                           (D) OTHER INFORMATION: - #/note= "Xho I restriction site."           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 9..44                                                  - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 27..44                                                          (D) OTHER INFORMATION: - #/note= "Region of homology with          target                                                                                         sequence."                                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                              - - CGACACTC GAG CTG CTG GAT CCG AAG CTC TGC TAC - #TTG CTA AAG                  - #44                                                                             Glu Leu Leu Asp Pro L - #ys Leu Cys Tyr Leu Leu Lys                            1        - #       5           - #       10                          - -  - - (2) INFORMATION FOR SEQ ID NO:30:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                              - - Glu Leu Leu Asp Pro Lys Leu Cys Tyr Leu Le - #u Lys                       1               5  - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:31:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 6..11                                                           (D) OTHER INFORMATION: - #/note= "Xho I restriction site."           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 12..31                                                          (D) OTHER INFORMATION: - #/note= "Region of homology with                          target se - #quence."                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 9..31                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                              - - CGACACTC GAG ACA ACA GAG TAC CAG GTA GC   - #                  - #              31                                                                              Glu Thr Thr Glu Tyr G - #ln Val Ala                                            1        - #       5                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:32:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                              - - Glu Thr Thr Glu Tyr Gln Val Ala                                           1               5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:33:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 6..11                                                           (D) OTHER INFORMATION: - #/note= "Xho I restriction site."           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 12..28                                                          (D) OTHER INFORMATION: - #/note= "Region of homology with                          target se - #quence."                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 9..28                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                              - - CGACACTC GAG GGC GTG CAG GTG GAG AC    - #                  - #                 28                                                                              Glu Gly Val Gln Val G - #lu Thr                                                1        - #       5                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:34:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                              - - Glu Gly Val Gln Val Glu Thr                                               1               5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:35:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 6..11                                                           (D) OTHER INFORMATION: - #/note= "Sal I restriction site."           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 12..27                                                          (D) OTHER INFORMATION: - #/note= "Region of homology with                          target se - #quence."                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: complement - #(9..26)                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                              - - CGACAGTCGA CTTCCAGTTT TAGAAGC          - #                  - #                 27                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:36:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                              - - Leu Leu Lys Leu Glu Val                                                   1               5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:37:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 7..12                                                           (D) OTHER INFORMATION: - #/note= "Xho I restriction site."           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 10..27                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 13..27                                                          (D) OTHER INFORMATION: - #/note= "Region of homology with                          target se - #quence."                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                              - - TCGACACTC GAG ACG GGG GCC GAG GGC      - #                  - #                 27                                                                               Glu Thr Gly Ala Glu - #Gly                                                     1       - #        5                                                - -  - - (2) INFORMATION FOR SEQ ID NO:38:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                              - - Glu Thr Gly Ala Glu Gly                                                    1               5                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:39:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 7..12                                                           (D) OTHER INFORMATION: - #/note= "Sal I restriction site."           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: complement - #(10..18)                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 13..28                                                          (D) OTHER INFORMATION: - #/note= "Region of homology with                          target se - #quence."                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                              - - CCGACAGTCG ACCTCTATTT TGAGCAGC         - #                  - #                 28                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:40:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                              - - Ile Glu Val                                                               1                                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:41:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                              - - CGACACCGCG GCCACCATGA AGCTACTGTC TTCTATCG      - #                      - #     38                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:42:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                              - - CGACAGTCGA CCGATACAGT CAACTGTC         - #                  - #                 28                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:43:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 6..11                                                           (D) OTHER INFORMATION: - #/note= "Sac II restriction site."          - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #signal                                           (B) LOCATION: 12..16                                                          (D) OTHER INFORMATION: - #/note= "Kozak sequence."                   - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 17..37                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 17..38                                                          (D) OTHER INFORMATION: - #/note= "Ga14 (1-147) coding region."       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                              - - CGACACCGCG GCCACC ATG AAG CTA CTG TCT TCT ATC - #G                      - #     38                                                                                       - #Met Lys Leu Leu Ser Ser Ile                                                - # 1               5                                        - -  - - (2) INFORMATION FOR SEQ ID NO:44:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                              - - Met Lys Leu Leu Ser Ser Ile                                               1               5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:45:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 1..17                                                           (D) OTHER INFORMATION: - #/note= "Region encoding for C-termina                    end of - #Ga14 (1-147)."                                        - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 3..17                                                  - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 18..23                                                          (D) OTHER INFORMATION: - #/note= "Sal I restriction site."           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                              - - GA CAG TTG ACT GTA TCG GTCGACTGTC G    - #                  - #                 28                                                                        Arg Gln Leu Thr Val Ser                                                        1              - # 5                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:46:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                              - - Arg Gln Leu Thr Val Ser                                                   1               5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:47:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                              - - CGACACCGCG GCCACCATGG TTTCTAAGCT GAGC       - #                  -      #        34                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:48:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                              - - CGACAGTCGA CCAACTTGTG CCGGAAGG         - #                  - #                 28                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:49:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 6..11                                                           (D) OTHER INFORMATION: - #/note= "Sac II restriction site."          - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #signal                                           (B) LOCATION: 12..16                                                          (D) OTHER INFORMATION: - #/note= "Kozak sequence."                   - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 17..34                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 17..34                                                          (D) OTHER INFORMATION: - #/note= "Region encoding N-terminal                       end of - #HNF1 (1281)."                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                              - - CGACACCGCG GCCACC ATG GTT TCT AAG CTG AGC   - #                  -      #        34                                                                                       - #Met Val Ser Lys Leu Ser                                                    - # 1               5                                        - -  - - (2) INFORMATION FOR SEQ ID NO:50:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                              - - Met Val Ser Lys Leu Ser                                                   1               5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:51:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 1..20                                                           (D) OTHER INFORMATION: - #/note= "Region encoding for             C-terminal                                                                                     end of - #HNF1 (1-282)."                                        - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 3..17                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                              - - CC TTC CGG CAC AAG TTG GTCGACTGTC G    - #                  - #                28                                                                     Ala Phe Arg His Lys Leu                                                        1               5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:52:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                              - - Ala Phe Arg His Lys Leu                                                   1               5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:53:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #signal                                           (B) LOCATION: 3..7                                                            (D) OTHER INFORMATION: - #/note= "Kozak sequence."                   - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 1..11                                                           (D) OTHER INFORMATION: - #/note= "Complementary to bases 5 to                      15 of - #SEQ ID NO:54."                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                              - - GGCCACCATG C               - #                  - #                      - #       11                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:54:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..3                                                            (D) OTHER INFORMATION: - #/note= "Translation product of SEQ                       ID NO:53 - #and SEQ ID NO:55.  Translational                                  start sit - #e at base 8 of SEQ ID NO:53."                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                              - - Met Leu Glu                                                               1                                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:55:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 14..17                                                          (D) OTHER INFORMATION: - #/note= "Sac II restriction site                          overhang."                                                      - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: - #/note= "Xho I restriction site                           overhang."                                                      - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 5..15                                                           (D) OTHER INFORMATION: - #/note= "Complementary to bases 1 to      11                                                                                             of SEQ - #ID NO:53."                                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                              - - TCGAGCATGG TGGCCGC             - #                  - #                      - #   17                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:56:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                              - - TCGACCCTAA GAMGAAGAGA AAGGTAC          - #                  - #                 27                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:57:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                              - - TCGAGTACCT TTCTCTTCKT CTTAGGG          - #                  - #                 27                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:58:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: - #/note= "Sal I restriction site           overhang."                                                                       - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 5..27                                                           (D) OTHER INFORMATION: - #/note= "Complementary to SEQ ID         NO:60,                                                                                         bases 5 - #to 27."                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                              - - TCGACCCTAA GAAGAAGAGA AAGGTAC          - #                  - #                 27                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:59:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..11                                                           (D) OTHER INFORMATION: - #/note= "Translation product of SEQ       ID                                                                                             NOS:58 an - #d 60."                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                              - - Leu Asp Pro Lys Lys Lys Arg Lys Val Leu Gl - #u                           1               5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO:60:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: - #/note= "Xho I restriction site                          overhang."                                                      - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 5..27                                                           (D) OTHER INFORMATION: - #/note= "Complementary to SEQ ID          NO:58,                                                                                         bases 5 - #to 27."                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                              - - TCGAGTACCT TTCTCTTCTT CTTAGGG          - #                  - #                 27                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:61:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                              - - CGACAGTCGA CGCCCCCCCG ACCGATGTC         - #                  - #                29                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:62:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                              - - CGACACTCGA GCCCACCGTA CTCGTC          - #                  - #                  26                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:63:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 6..11                                                           (D) OTHER INFORMATION: - #/note= "Sal I restriction site."           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 12..29                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 12..29                                                          (D) OTHER INFORMATION: - #/note= "Region encoding Nterminal                        end of - #VP16 (413490)."                                       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                              - - CGACAGTCGA C GCC CCC CCG ACC GAT GTC    - #                  - #                29                                                                                  Ala Pro Pro - #Thr Asp Val                                                      1   - #            5                                            - -  - - (2) INFORMATION FOR SEQ ID NO:64:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                              - - Ala Pro Pro Thr Asp Val                                                    1               5                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:65:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..15                                                  - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 1..15                                                           (D) OTHER INFORMATION: - #/note= "Region encoding C-terminal                       end of - #VP16 (413-490)."                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                              - - GAC GAG TAC GGT GGG CTCGAGTGTC G      - #                  - #                  26                                                                     Asp Glu Tyr Gly Gly                                                            1               5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:66:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:                              - - Asp Glu Tyr Gly Gly                                                       1               5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:67:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:                              - - GGAATTCCAT ATGGGCGTGC AGG           - #                  - #                  23                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:68:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:                              - - His Met Gly Val Gln                                                        1               5                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:69:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:                              - - CTGTCCCGGG ANNNNNNNNN TTTCTTTCCA TCTTCAAGC      - #                      - #  39                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:70:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:                              - - Arg Ser Xaa Xaa Xaa Lys Lys Gly Asp Glu Le - #u                            1               5 - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO:71:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 64 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:                              - - CTGTCCCGGG AGGAATCAAA TTTCTTTCCA TCTTCAAGCA TNNNNNNNNN  - #GTGCACCAC    G   60                                                                          - - CAGG                 - #                  - #                  - #                64                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:72:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  19 amin - #o acids                                               (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:                              - - Arg Ser Ser Asp Phe Lys Lys Gly Asp Glu Le - #u Met Xaa Xaa Xaa His       1               5  - #                10  - #                15               - - Val Val Cys                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:73:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 57 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:                              - - CGCGGATCCT CATTCCAGTT TTAGAAGCTC CACATCNNNN NNNNNAGTGG CA - #TGTGG          57                                                                          - -  - - (2) INFORMATION FOR SEQ ID NO:74:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:                              - - Glu Leu Lys Leu Leu Glu Val Asp Xaa Xaa Xa - #a Thr Ala His Pro           1               5  - #                10  - #                15               - -  - - (2) INFORMATION FOR SEQ ID NO:75:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:                              - - CGCGGATCCT CATTCCAGTT TTAGAAGC         - #                  - #               28                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:76:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:                              - - Glu Leu Lys Leu Leu                                                        1               5                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:77:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:                              - - CGACAGTCGA CCGATACAGT CAACTGTC         - #                  - #              28                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:78:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:                              - - CGACAGTCGA CCAACTTGTG CCGGAAGG         - #                  - #               28                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:79:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:                              - - TCGAGCATGG TGGCCGC             - #                  - #                      - #  17                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:80:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:                              - - TCGAGTACCT TTCTCTTCTT CTTAGGG          - #                  - #                 27                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:81:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:                              - - CGACACTCGA GCCCACCGTA CTCGTC          - #                  - #                  26                                                                    __________________________________________________________________________

We claim:
 1. A composition comprising two genetic constructs encodingfirst and second chimeric proteins,(i) the first chimeric proteincomprising (a) at least one ligand-binding domain which binds to aselected ligand to form a ligand cross-linked protein complex includingthe first and second chimeric proteins, and (b) an action domain whichis heterologous to said at least one ligand binding domain and whichinduces a biological process as a result of formation of the ligandcross-linked protein complex; and (ii) the second chimeric proteincomprising (a) at least one ligand binding domain which binds to aselected ligand, which ligand binding domain may be the same ordifferent from a ligand binding domain of the first chimeric protein,and (b) an intracellular targeting domain which is heterologous withrespect to said at least one ligand binding domain and causes cellularlocalization of the ligand-cross-linked protein complexes.
 2. Thecomposition according to claim 1, wherein the intracellular targetingdomain comprises a secretory leader sequence, a membrane retentiondomain, a nuclear localization domain or a vesicle targeting domain. 3.The composition according to claim 1, wherein the intracellulartargeting domain includes a membrane retention domain comprising aplasma membrane targeting sequence for attachment of a myristoyl moietyor prenyl moiety.
 4. The composition according to claim 1, wherein theintracellular targeting domain includes a membrane retention domaincomprising a transmembrane domain.
 5. The composition according to claim1, wherein the intracellular targeting domain includes a nuclearlocalization domain comprising a bipartite basic repeat.
 6. Thecomposition according to claim 1, wherein at least one of theligand-binding domains comprises a ligand-binding domain of a steroidreceptor.
 7. The composition according to claim 1, wherein at least oneof the ligand-binding domain comprises an antibiotic binding domain. 8.The composition according to claim 1, wherein at least one of theligand-binding domains comprises a ligand-binding domain of atetracycline receptor.
 9. The composition according to claim 1, whereinat least one of the ligand-binding domains comprises an antibody domain.10. The composition according to claim 1, wherein at least one of theligand-binding domains comprises a ligand-binding domain of acyclophilin receptor.
 11. The composition according to claim 1, whereinat least one of the ligand-binding domains comprises a ligand bindingdomain of an immunophilin.
 12. The composition according to claim 1,wherein at least one of the ligand-binding domains binds to FK506,FK520, rapamycin or a derivative of any of the foregoing.
 13. Thecomposition according to claim 1, wherein at least one of theligand-binding domains comprises an FK506 binding domain of a humanFK506 Binding Protein (FKBP12) or a variant thereof in which one or moreof Tyr26, Phe36, Asp37, Tyr82 and Phe99 are replaced by other amino acidresidues.
 14. The composition according to claim 1, wherein at least oneof the chimeric proteins includes two or more ligand-binding domains.15. The composition according to claim 1, wherein the ligand-bindingdomains are from 50 to 350 amino acid residues in length.
 16. Thecomposition according to claim 1, wherein at least one of theligand-binding domains comprises a naturally-occurring peptide sequence.17. The composition according to claim 1, wherein at least one of theligand-binding domains comprises a non-naturally-occurring peptidesequence.
 18. The composition according to claim 1, further includingthe ligand formulated in an amount sufficient to induce the biologicalprocess as a result of formation of the ligand cross-linked proteincomplex.
 19. The composition according to claim 1, wherein the actiondomain comprises:(a) a DNA binding domain; (b) a transcriptionalactivation domain; or (c) a transcriptional repressor domain.
 20. Thecomposition according to claim 1, wherein the biological processincludes a detectable protein kinase activity.
 21. The compositionaccording to claim 1, wherein the biological process includes adetectable phosphatase activity.
 22. The composition according to claim1, wherein the biological process includes a detectable activitycomprising reductase activity, cyclooxygenase activity or proteaseactivity.
 23. The composition according to claim 1, wherein the actiondomain includes a cytoplasmic domain of a cell surface receptor, or avariant thereof sufficient to induce the biological process in the cellas a result of formation of the ligand cross-linked protein complex. 24.The composition according to claim 1, wherein the biological process isselected from the group consisting of channel opening, ion release,acylation, methylation, hydrolysis, phosphorylation, dephosphorylation,change in redox states, and rearrangement reactions.
 25. The compositionaccording to claim 1, wherein the biological process includes celldeath.
 26. The composition according to claim 1, wherein the biologicalprocess includes regulation of gene transcription.
 27. The compositionaccording to claim 11, wherein the ligand-binding domain comprises aligand-binding domain of an FK506 Binding Protein.
 28. The compositionaccording to claim 11, wherein the ligand-binding domain is a ligandbinding domain of a human FK506 Binding Protein.
 29. The compositionaccording to claim 13, wherein one or more of Tyr26, Phe36, Asp37, Tyr82and Phe99 of the FKBP 12 are replaced by amino acids independentlyselected from the group consisting of Val, Ala, Gly and Met.
 30. Thecomposition according to claim 13, wherein one or both of Phe36 andAsp37 are replaced by amino acids independently selected from the groupconsisting of Val and Ala.
 31. The composition according to claim 13,wherein Phe36 is replaced by a valine residue.
 32. The compositionaccording to claim 13, wherein Phe36 is replaced by a methionineresidue.
 33. The composition according to claim 14, wherein two or moreof the ligand-binding domains have different ligand bindingspecificities.
 34. The composition according to claim 15, wherein theligand-binding domains are from 50 to 200 amino acid residues in length.35. The composition according to claim 17, wherein thenon-naturally-occurring ligand-binding domain differs from anaturally-occurring ligand binding domain by one or more amino acidresidues, and binds a ligand which is not bound by thenaturally-occurring ligand binding domain.
 36. The composition accordingto claim 23, wherein the action domain is derived from the cytoplasmicdomain of a cell surface receptor selected-from the group consisting ofa tyrosine kinase receptor, a cytokine receptor and a growth factorreceptor.
 37. The composition according to claim 23, wherein thereceptor is selected from the group consisting of CD3ζ, CD3η, CD3γ,CD3δ, CD3ε, an interferon receptor, an interleukin receptor, a GM-CSFreceptor, a LIF receptor, a CNTF receptor, an oncostatin M receptor, aTGF-β receptor, an EGF receptor, ATR2/neu, a HER2/neu, a HER3/c-erbB-3,Xmrk, an insulin receptor, an IGF-1 receptor, IRR, PDGF receptor, aCSF-I receptor, c-kit, STK-1/flk-2, an FGF receptor, flg, bek, an NGFreceptor, Ig-alpha/MB-1, Ig-beta/B29, Ror1 and Ror2.
 38. The compositionaccording to any of claim 1, 8 or 24, further comprising a heterologoustarget gene under the expression control of a transcriptional controlelement responsive to formation of the ligand cross-linked complex. 39.The composition according to claim 25, wherein the action domaincomprises a cytoplasmic portion of a Fas or TNF receptor sufficient toinduce cell death as a result of formation of the ligand cross-linkedprotein complex.
 40. The composition according to claim 26, wherein thebiological process includes regulation of a gene having atranscriptional regulatory element selected from the group consisting ofa cAMP responsive element, an SRE, a VL30, an RSRF, an ISRE, a GAS, anARRE-1 and an ARRE-2.
 41. The composition according to claim 26, whereinthe biological process includes regulation of expression of anendogenous gene of the cell.
 42. The composition according to claim 26,wherein the biological process includes regulation of expression of aheterologous gene included in the cell.
 43. The composition of claim 38,wherein the target gene encodes a surface membrane protein, a secretedprotein, a cytoplasmic protein, an antisense messsage or a ribozyme. 44.The composition of claim 38, wherein the target gene encodes a hormone,growth factor, interleukin, enzyme or surface membrane protein.
 45. Thecomposition according to any of claims 1, 2, 13, 17-19, 23, 25-27, 31,41 or 42, wherein the ligand-binding domains bind the ligand with a Kdless than or equal to 10⁻⁶ M.
 46. The composition according to any ofclaims 1, 2, 13, 17-19, 23, 25-27, 31, 41 or 42, wherein theligand-binding domains are provided in the chimeric proteins asintracellular domains.
 47. The composition according to any of claims 1,2, 13, 17-19, 23, 25-27, 31, 41 or 42, wherein at least one of theligand-binding domains comprises a ligand-binding domain of anintracellular protein.
 48. The composition according to any of claims 1,2, 13, 17-19, 23, 25-27, 31, 41 or 42, wherein the ligand-bindingdomains bind a ligand which is a synthetic organic molecule having amolecular weight of less than 5 kD.
 49. The composition according to anyof claims 1, 2, 13, 17-19, 23, 25-27, 31, 41 or 42, wherein theligand-binding domains bind a ligand which is membrane permeable. 50.The composition according to any of claims 1, 2, 13, 17-19, 23, 25-27,31, 41 or 42, wherein the ligand-binding domains bind a ligand which isnot a protein.
 51. The composition according to any of claims 1-14, 18,33, 35-37, 39, 41 or 42, wherein the ligand cross-linked protein complexcomprises two or more chimeric proteins.
 52. The composition accordingto any of claims 1-14, 18, 33, 35-37, 39, 41 or 42, wherein each of thegenetic constructs is provided in a vector which further comprises oneor more of (i) an origin of replication, (ii) a selectable marker, (iii)an amplifiable marker, and (iv) a transcriptional regulatory sequencefor expressing the chimeric proteins in mammalian cells.
 53. Thecomposition according to claim 45, wherein the ligand-binding domainsbind the ligand with a Kd less than or equal to 10⁻⁸ M.
 54. Thecomposition according to claim 45, wherein the ligand is membranepermeable and the ligand-binding domains are provided in the chimericproteins as intracellular domains.
 55. The composition according toclaim 50, wherein the ligand-binding domains bind a ligand which is amacrocyclic compound.
 56. The composition according to claim 52, whereinat least one of the vectors is a viral vector.
 57. The compositionaccording to claim 52, wherein the constructs are provided on separatevectors.
 58. The composition according to claim 52, wherein theconstructs are provided on the same vector.
 59. The compositionaccording to claim 52, wherein each of the the genetic constructsincludes a transcriptional regulatory sequence for expressing thechimeric protein in human cells.
 60. The composition according to claim54, wherein the ligand-binding domains bind a ligand which is asynthetic organic molecule having a molecular weight of less than 5 kD.61. The composition according to claim 54, wherein the ligandcross-linked protein complex comprises two or more chimeric proteins.62. The composition according to claim 55, wherein the macrocycliccompound comprises a macrolide.
 63. The composition according to claim56, wherein the viral vector is an adenoviral vector.
 64. Thecomposition according to claim 56, wherein the viral vector is anadeno-associated viral vector.
 65. The composition according to claim56, wherein the viral vector is a Herpes simplex viral vector.
 66. Thecomposition according to claim 56, wherein the viral vector is aretroviral vector.
 67. The composition according to claim 60, whereinthe ligand-binding domains bind a ligand which is not a protein.
 68. Acomposition comprising (a) two genetic constructs each encoding adifferent chimeric protein which binds to a common selected ligand toform an oligomeric ligand-cross-linked complex including both chimericproteins, and (b) an additional genetic construct comprising a targetgene under the transcriptional control of a transcriptional controlelement responsive to the formation of the ligand cross-linked proteincomplex.
 69. The composition of claim 68, wherein one of the chimericproteins comprises a transcriptional activation domain, the otherchimeric protein comprises a DNA-binding domain, and the transcriptionalcontrol element binds the DNA-binding domain.
 70. The composition ofclaim 68, wherein the target gene encodes a surface membrane protein, asecreted protein, a cytoplasmic protein, an antisense message or aribozyme.
 71. The composition of claim 68, wherein the target geneencodes a hormone, growth factor, interleukin, enzyme or surfacemembrane protein.